Non-protein clostridial toxin compositions

ABSTRACT

Pharmaceutical compositions that stabilize a Clostridial toxin active ingredient are described. The compositions can be liquid or solid compositions, and comprise a surfactant and an antioxidant. In some embodiments, the compositions comprise a surfactant selected from a poloxamer and a polysorbate; an antioxidant selected from methionine, N-acetyl cysteine, ethylenediaminetetraacetic acid and combinations thereof, and, optionally, a tonicity agent and/or a lyoprotector selected from, for example, trehalose, sucrose.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/394,009, filed Sep. 13, 2016, incorporated by reference herein in itsentirety.

TECHNICAL FIELD

The present disclosure relates to solid and liquid pharmaceuticalcompositions comprising a Clostridial toxin active ingredient and one ormore non-protein excipient.

BACKGROUND

A pharmaceutical composition is a formulation which contains at leastone active ingredient (such as a Clostridial toxin) as well as, forexample, one or more excipients, buffers, carriers, stabilizers,preservatives and/or bulking agents, and is suitable for administrationto a patient to achieve a desired diagnostic result or therapeuticeffect. The pharmaceutical compositions disclosed herein havediagnostic, therapeutic and/or research utility.

For storage stability and convenience of handling, a pharmaceuticalcomposition can be formulated as a lyophilized (i.e. freeze dried) orvacuum dried powder which can be reconstituted with a suitable fluid,such as saline or water, prior to administration to a patient.Alternately, the pharmaceutical composition can be formulated as anaqueous solution or suspension. A pharmaceutical composition can containa proteinaceous active ingredient. Unfortunately, a protein activeingredient can be very difficult to stabilize (i.e. maintained in astate where loss of biological activity is minimized), thereby resultingin a loss of protein and/or loss of protein activity during theformulation, reconstitution (if required) and storage of thepharmaceutical composition prior to use. Stability problems can arisedue to surface adsorption of a protein active ingredient, physicalinstability, such as, e.g., denaturation or aggregation, or chemicalinstability, such as, e.g., cross-linking, deamidation, isomerization,oxidation, formation of acidic or basic species, Maillard reaction, andfragmentation. To prevent such instability, various protein-basedexcipients, such as albumin and gelatin, have been used to stabilize aprotein active ingredient present in a pharmaceutical composition.

Unfortunately, despite their known stabilizing effects, significantdrawbacks exist to the use of protein excipients, such as albumin orgelatin, in a pharmaceutical composition. For example, albumin andgelatin are expensive and increasingly difficult to obtain. Furthermore,blood products or animal derived products such as albumin and gelatin,when administered to a patient can subject the patient to a potentialrisk of receiving blood borne pathogens or infectious agents. Thus, itis known that the possibility exists that the presence of ananimal-derived protein excipient in a pharmaceutical composition canresult in inadvertent incorporation of infectious elements into thepharmaceutical composition. For example, it has been reported that useof human serum albumin may transmit prions into a pharmaceuticalcomposition. Thus, it is desirable to find suitable non-proteinexcipients, such as, e.g., stabilizers, cryo-protectants andlyoprotectants, which can be used to stabilize the protein activeingredient present in a pharmaceutical composition.

The unique characteristics of Clostridial toxins further constrain andhinder the selection of suitable non-protein excipients for apharmaceutical composition comprising a Clostridial toxin. For example,Clostridial toxins are large proteins having an average molecular weightof approximately 150 kDa, and are further complexed with non-toxinassociated proteins that increase the size to approximately 300-900-kDa.The size of a Clostridial toxin complex makes it much more fragile andlabile than smaller, less complex proteins, thereby compounding theformulation and handling difficulties if Clostridial toxin stability isto be maintained. Hence, the use of non-protein excipients, such as,e.g., stabilizers, cryo-protectants and lyoprotectants must be able tointeract with the Clostridial toxin in a manner which does not denature,fragment or otherwise inactivate the toxin or cause disassociation ofthe non-toxin associated proteins present in the toxin complex.

Another problem associated with a Clostridial toxin, is the exceptionalsafety, precision, and accuracy that is necessary for at all steps ofthe formulation process. Thus, a non-protein excipient should not itselfbe toxic or difficult to handle so as to not exacerbate the alreadyextremely stringent requirements.

Still another difficulty linked with a Clostridial toxin, is the smallamount of Clostridial toxin that is used in a pharmaceuticalcomposition. As with enzymes generally, the biological activities of theClostridial toxins are dependent, at least in part, upon theirthree-dimensional conformation. Thus, a Clostridial toxin is detoxifiedby heat, various chemicals, surface stretching, and surface drying.Additionally, it is known that dilution of a Clostridial toxin complexobtained by the known culturing, fermentation and purification methodsto the much lower concentration used in a pharmaceutical compositionresults in rapid inactivation of the toxin. The low amount of aClostridial toxin that is used in a pharmaceutical composition, makesthis active ingredient very susceptible to adsorption to, e.g., thesurfaces of laboratory glassware, vessels, to the vial in which thepharmaceutical composition is reconstituted and to the inside surface ofa syringe used to inject the pharmaceutical composition. Such adsorptionof a Clostridial toxin to surfaces can lead to a loss of activeingredient and to denaturation of the remaining Clostridial toxin, bothof which reduce the total activity of the active ingredient present inthe pharmaceutical composition. Hence, the use of non-proteinexcipients, such as, e.g., stabilizers, cryo-protectants andlyoprotectants must be able to act as surface blockers to prevent theadsorption of a Clostridial toxin to a surface.

Yet another problem connected to a Clostridial toxin, is thepH-sensitivity associated with complex formation. For example, the900-kDa BoNT/A complex is known to be soluble in dilute aqueoussolutions at pH 3.5-6.8. However, at a pH above about 7 the non-toxicassociated proteins dissociate from the 150-kDa neurotoxin, resulting ina loss of toxicity, particularly as the pH rises above pH 8.0. SeeEdward J. Schantz et al., pp. 44-45, Preparation and characterization ofbotulinum toxin type A for human treatment, in Jankovic, J., et al.,Therapy with Botulinum Toxin (Marcel Dekker, Inc., 1994). As thenon-toxic associated proteins are believed to preserve or help stabilizethe secondary and tertiary structures upon which toxicity depends, thedissociation of these proteins results in a more unstable Clostridialtoxin. Thus, non-protein excipients useful to formulate a pharmaceuticalcomposition comprising a Clostridial toxin must be able to operatewithin the confines of a pH level necessary to maintain the activity aClostridial toxin.

What is needed therefore is a Clostridial toxin pharmaceuticalcomposition wherein a Clostridial toxin active ingredient (such as abotulinum toxin) is stabilized by a non-protein excipient. The presentcompositions relate to solid and liquid Clostridial toxin activeingredient pharmaceutical compositions with one or more non-proteinexcipients which functions to stabilize the Clostridial toxin activeingredient present in the solid or liquid pharmaceutical composition.

BRIEF SUMMARY

In one aspect, a pharmaceutical composition comprising a Clostridialtoxin active ingredient, a tonicity agent, a surfactant and anantioxidant is provided.

In another aspect, a pharmaceutical composition comprising a Clostridialtoxin active ingredient, a surfactant and an antioxidant is provided.

In another aspect, a pharmaceutical composition comprising a Clostridialtoxin active ingredient, a lyoprotector, a surfactant and an antioxidantis provided.

In some embodiments, the pharmaceutical compositions comprise abotulinum toxin. In some embodiments, the pharmaceutical compositioncomprises trehalose. In some embodiments, the pharmaceutical compositioncomprises sodium chloride. In some embodiments, the compositioncomprises a poloxamer and/or a polysorbate. In some embodiments, thecomposition comprises poloxamer 188 and/or polysorbate 20. In someembodiments, the antioxidant is selected from the group consisting ofL-methionine, N-acetyl-cysteine (NAC), butylated hydroxytoluene (BHT),ethylene diamine tetraacetic acid sodium salt (EDTA), an EDTA analog,ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA), an EGTA analog, diethylenetriaminepentaacetic acid (DTPA), aDTPA analog, ascorbic acid, and combinations thereof. In someembodiments, the composition further comprises a buffering agent. In oneembodiment, the buffering agent includes histidine buffer. In someembodiments, the composition has a pH of from 5 to 7. In someembodiments, the composition is a liquid formulation. In someembodiments, the composition is a solid lyophilized formulation.

In another aspect, a liquid pharmaceutical composition comprising aClostridial toxin active ingredient, trehalose, a poloxamer or apolysorbate, and L-methionine or NAC is provided. In some embodiments,the liquid pharmaceutical composition comprises a botulinum toxin. Insome embodiments, the liquid pharmaceutical composition furthercomprises EDTA, EGTA, DTPA or analogs thereof. In some embodiments, theliquid pharmaceutical composition comprises a histidine buffer. In someembodiments, the pH of the liquid pharmaceutical composition ranges from5 to 7. In some embodiments, the relative weight amount of L-methionineranges from about 0.1% to about 0.3%. In some embodiments, the relativeweight amount of NAC ranges from about 0.1% to about 0.5%. In someembodiments, the relative weight amount of EDTA ranges from about 0.01%to about 0.05%. In some embodiments, the relative weight amount oftrehalose ranges from about 1.0 to about 10%. In some embodiments, therelative weight amount of poloxamer 188 ranges from about 0.5% to about5%. In some embodiments, the relative weight amount of polysorbateranges from about 0.02% to about 0.06%.

In another embodiment, a liquid pharmaceutical composition is provided.The composition comprises a Clostridial toxin active ingredient; atonicity agent selected from trehalose, sucrose and combinationsthereof; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof; and an antioxidant selected from methionine, NAC,ascorbic acid, butylated hydroxytoluene, EDTA, EGTA, DTPA, analogsthereof, and combinations thereof. In one embodiment, the compositionexcludes albumin, and in another embodiment, when the antioxidant ismethionine the composition excludes a polysorbate. In one embodiment,the composition excludes animal protein stabilizers.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof and an antioxidant selected from methionine, NAC,EDTA, EGTA, DTPA, analogs thereof, and combinations thereof. In oneembodiment, the composition excludes albumin.

In another embodiment, the liquid composition is an animal protein freecomposition that comprises a botulinum toxin, a poloxamer, andmethionine, and optionally includes a disaccharide. In one embodiment,the liquid composition excludes a disaccharide.

In a further embodiment, the liquid composition is an animal proteinfree composition that comprises a botulinum toxin, a surfactant selectedfrom a poloxamer, a polysorbate and combinations thereof and anantioxidant selected from the group consisting of a chelating agent, asacrificial antioxidant, a chain terminator, and combinations thereof.In one embodiment, the antioxidant includes a combination of a chelatingagent and a chain terminator.

In yet another embodiment, the liquid composition is an animal-proteinfree composition comprising a botulinum toxin, a poloxamer surfactant,and methionine, and optionally a disaccharide. In one embodiment, theliquid composition excludes a disaccharide.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; trehalose or sucrose; a poloxamer; and methionine. In oneembodiment, the composition excludes albumin.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient, trehalose in an amount between 1-15 wt %; a poloxamer in anamount between 0.5-8 wt %; and methionine in an amount between 0.05-5 wt%. In one embodiment, the composition excludes albumin.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a botulinum toxin; adisaccharide; a poloxamer; and an antioxidant selected from methionine,NAC, ascorbic acid, butylated hydroxytoluene, EDTA, EGTA, DTPA, analogsthereof, and combinations thereof. In one embodiment, the compositionexcludes albumin.

In another embodiment, the liquid composition is an animal protein freecomposition that comprises a botulinum toxin; a disaccharide; apoloxamer; and an antioxidant selected from the group consisting of achelating agent, a sacrificial antioxidant, a chain terminator, andcombinations thereof.

In another embodiment, a liquid composition comprised of a Clostridialtoxin active ingredient, a poloxamer, a chelating agent and a chainterminator is provided.

In another embodiment, a liquid composition comprised of a Clostridialtoxin active ingredient; a poloxamer; a chelating agent selected fromEDTA, EGTA, DTPA and analogs thereof; and NAC is provided.

In another embodiment, a liquid composition comprised of a Clostridialtoxin active ingredient, a poloxamer, and methionine is provided.

In another embodiment, a liquid composition comprised of a Clostridialtoxin active ingredient, a tonicity agent selected from trehalose,sucrose and combinations thereof, a surfactant selected from apoloxamer, a polysorbate and combinations thereof, a chelating agent anda chain terminator is provided.

In another embodiment, a liquid composition comprised of a Clostridialtoxin active ingredient; a tonicity agent selected from trehalose,sucrose and combinations thereof; a surfactant selected from apoloxamer, a polysorbate and combinations thereof; a chelating agentselected from EDTA, EGTA, DTPA and analogs thereof; and NAC, isprovided.

In any of the foregoing embodiments, it is contemplated that thecomposition is not, in some embodiments, an emulsion and/or excludesnanoparticles comprising an amphiphilic entity.

In another aspect, the present disclosure provides a solidpharmaceutical composition comprising a botulinum toxin; trehalose; apoloxamer or a polysorbate; NAC; and a chelating agent selected fromEDTA, EGTA, DTPA and analogs thereof. In an alternative embodiment, thesolid pharmaceutical composition comprises a botulinum toxin, trehalose,a poloxamer and L-methionine. In some embodiments, the solidpharmaceutical composition further comprises histidine buffer. In someembodiments, the relative weight amount of L-methionine ranges fromabout 0.1% to about 0.3%. In some embodiments, the relative weightamount of NAC ranges from about 0.01% to about 0.5%. In someembodiments, the relative weight amount of EDTA ranges from about 0.01%to about 0.05%. In some embodiments, the relative weight amount oftrehalose ranges from about 1.0 to about 10%. In some embodiments, therelative weight amount of poloxamer ranges from about 0.5% to about 5%.In some embodiments, the relative weight amount of polysorbate rangesfrom about 0.02% to about 0.06%.

In one embodiment, a solid or lyophilized pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a disaccharide selected from trehalose, sucrose andcombinations thereof; a surfactant selected from a poloxamer, apolysorbate and combinations thereof and an antioxidant selected frommethionine, N-acetyl cysteine, BHT, EDTA, EGTA, DTPA, ascorbic acid,analogs thereof, and combinations thereof. In one embodiment, thecomposition excludes albumin, a hydroxyalkyl starch, glutamic acid,glutamine, aspartic acid, asparagine, a polyalcohol, glycine, and/orpolyvinylpyrrolidone.

In another embodiment, the solid or lyophilized composition is an animalprotein free composition that comprises a botulinum toxin; adisaccharide selected from trehalose, sucrose and combinations thereof;a surfactant selected from a poloxamer, a polysorbate and combinationsthereof and an antioxidant selected from the group consisting of achelating agent, a sacrificial antioxidant, a chain terminator, andcombinations thereof.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a Clostridial toxin activeingredient; trehalose or sucrose; a poloxamer; and methionine. In oneembodiment, the composition excludes albumin, a hydroxyalkyl starch,glutamic acid, glutamine, aspartic acid, asparagine, a polyalcohol,glycine, and/or polyvinylpyrrolidone.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a Clostridial toxin activeingredient; trehalose in an amount between 1-15 wt %; a poloxamer in anamount between 0.5-8 wt %; and methionine in an amount between 0.05-5 wt%. In one embodiment, the composition excludes albumin, a hydroxyalkylstarch, glutamic acid, glutamine, aspartic acid, asparagine, apolyalcohol, glycine, and/or polyvinylpyrrolidone.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a botulinum toxin; adisaccharide; a poloxamer; and an antioxidant selected from methionine,N-acetyl cysteine, EDTA, EGTA, DTPA, analogs thereof, and combinationsthereof. In one embodiment, the composition excludes albumin, ahydroxyalkyl starch, glutamic acid, glutamine, aspartic acid,asparagine, a polyalcohol, glycine, and/or polyvinylpyrrolidone.

In another embodiment, the solid or lyophilized composition is an animalprotein free composition that comprises a botulinum toxin, adisaccharide, a poloxamer; and an antioxidant selected from the groupconsisting of a chelating agent, a sacrificial antioxidant, a chainterminator, and combinations thereof.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof; a chelating agent; and a chain terminator; isprovided.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof; a chelating agent selected from EDTA, EGTA, DTPA,and analogs thereof; and NAC; is provided.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a poloxamer; and a chain terminator is provided. In oneembodiment, the lyophilized composition excludes a chelating agent. Inone embodiment, the chain terminator is NAC.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a poloxamer; and methionine is provided.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a poloxamer; a chelating agent; and a chain terminator isprovided.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a poloxamer; a chelating agent selected from EDTA, EGTA, DTPAand analogs thereof; and NAC is provided.

In another embodiment, the lyophilized composition is an animal proteinfree composition that comprises a Clostridial toxin active ingredient; alyoprotector selected from sucrose, trehalose, mannitol, sorbitol,glucose, and combinations thereof; a poloxamer; and NAC; and optionallyincludes EDTA, EGTA, DTPA or analogs thereof. In one embodiment, thelyophilized composition excludes EDTA, EGTA, DTPA and analogs thereof.

In another embodiment, a lyophilized composition comprised of aClostridial toxin active ingredient; a lyoprotector selected fromsucrose, trehalose, mannitol, sorbitol, glucose, and combinationsthereof; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof; and NAC is provided.

In certain embodiments, the lyophilized composition is reconstitutedwith a tonicity agent selected from trehalose, sucrose, sodium chloride,mannitol, sorbitol, glucose, and combinations thereof. In at least oneembodiment, the lyophilized composition is reconstituted with areconstitution vehicle comprising NaCl prior to administration to apatient.

In any of the foregoing embodiments of solid or liquid compositions, itis contemplated that one or more, in any combination, of theseingredients are excluded polyvinylpyrrolidone, diblock copolymers ofpolypropylene glycol and polyethylene glycol, and/or a polyalcohol suchas inositol, lactilol, isomalt, xylitol, erythritol. In any of theforegoing embodiments of solid or liquid compositions, it iscontemplated that the composition is free of animal proteins.

In another aspect, a pharmaceutical composition comprising a Clostridialtoxin active ingredient; a tonicity agent and/or a lyoprotector selectedfrom trehalose, sucrose and combinations thereof a surfactant selectedfrom a poloxamer, a polysorbate and combinations thereof; and anantioxidant selected from methionine, N-acetyl cysteine, EDTA, EGTA,DTPA, analogs thereof, and combinations thereof, is contemplated. In oneembodiment, the composition excludes albumin, and in embodiments whenthe composition is a liquid and the antioxidant is methionine, thesurfactant excludes a polysorbate. The composition can be liquid orsolid.

In another aspect, a pharmaceutical composition is contemplated. Thecomposition comprises a Clostridial toxin active ingredient; trehaloseor sucrose; a poloxamer; and methionine. In one embodiment, thecomposition excludes albumin. The composition can be liquid or solid.

In another aspect, a pharmaceutical composition is contemplated. Thecomposition comprises a Clostridial toxin active ingredient, trehalosein an amount between 1-15 wt %, a poloxamer in an amount between 0.5-8wt %, and methionine in an amount between 0.05-5 wt %. In oneembodiment, the composition excludes albumin. The composition can beliquid or solid.

In another aspect, a pharmaceutical composition is contemplated. Thecomposition comprises a botulinum toxin; a disaccharide; a poloxamer;and an antioxidant selected from methionine, NAC, EDTA, EGTA, DTPA,analogs thereof, and combinations thereof. In one embodiment, thecomposition excludes albumin. The composition can be liquid or solid.

In another aspect, a pharmaceutical composition comprising a Clostridialtoxin active ingredient; a surfactant selected from a poloxamer, apolysorbate and combinations thereof; and an antioxidant selected frommethionine, N-acetyl cysteine, EDTA, EGTA, DTPA, analogs thereof, andcombinations thereof, is provided. In one embodiment, the compositionexcludes albumin, and in embodiments when the composition is a liquidand the antioxidant is methionine, the surfactant excludes apolysorbate. The composition can be liquid or solid. In one embodiment,the composition further comprises a tonicity agent and/or alyoprotector. In some embodiments, the tonicity agent is selected fromtrehalose, sucrose, sodium chloride, mannitol, sorbitol, glucose, andcombinations thereof. In alternative embodiments, the lyoprotector isselected from trehalose, sucrose, mannitol, sorbitol, glucose, andcombinations thereof. In one embodiment, the tonicity agent and/orlyoprotector is a disaccharide. In one embodiment, the disaccharide isselected from trehalose and sucrose.

In another aspect, a method for treating depression is contemplated. Themethod comprises providing for administration, instructing toadminister, or administering a composition according to any of theembodiments and aspects described herein.

In another aspect, a method for treating cardiac arrhythmia iscontemplated. The method comprises providing for administration,instructing to administer, or administering a composition according toany of the embodiments and aspects described herein.

DETAILED DESCRIPTION

Compositions described herein, in embodiments, are directed to stableliquid and/or stable solid pharmaceutical compositions of a Clostridialtoxin active ingredient, where the composition comprises, in someembodiments, a surfactant and an antioxidant, and optionally a tonicityagent and/or a lyoprotector. In certain liquid compositions, thedisaccharide is optional.

Also as will be described below, the compositions are useful in methodsfor the treatment of various diseases, disorders, and conditions,including, for example, depression (e.g. major depressive disorder),headache (e.g. migraine, tension headache, and the like), pain, atrialfibrillation, hyperhidrosis, muscle spasticity, cervical dystonia,blepherospasm, overactive bladder (e.g. neurogenic detrusorover-activity, and idiopathic overactive bladder), bladder pain (e.g.interstitial cystitis, and bladder pain syndrome), skin conditions (e.g.wrinkles, fine wrinkles, excess sebum production, acne, and rosacea),irregularities, and the like using the compositions provided herein.Embodiments can include various administration techniques, including,for example, injection, such as intramuscular, intracutaneous,subcutaneous, or the like, instillation, intravenous, transdermal, andtopical.

Definitions

As used herein, the words or terms set forth below have the followingdefinitions:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” or “approximately” as used herein means within an acceptableerror range for the particular value as determined by one of ordinaryskill in the art, which will depend in part on how the value is measuredor determined, (i.e., the limitations of the measurement system). Forexample, “about” can mean within 1 or more than 1 standard deviations,per practice in the art. Where particular values are described in theapplication and claims, unless otherwise stated, the term “about” meanswithin an acceptable error range for the particular value. The term“about” when qualifying a value of a stated item, number, percentage, orterm refers to a range of plus or minus ten percent of the value of thestated item, percentage, parameter, or term.

“Administration”, or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition to a subject, oralternatively a subject receiving a pharmaceutical composition. Thepharmaceutical compositions disclosed herein can be locally administeredby various methods. For example, intramuscular, intradermal,subcutaneous administration, intrathecal administration, intraperitonealadministration, topical (transdermal), instillation, and implantation(for example, of a slow-release device such as polymeric implant orminiosmotic pump) can all be appropriate routes of administration.

“Alleviating” means a reduction in the occurrence of a pain, of aheadache, or of any symptom or cause of a condition or disorder. Thus,alleviating includes some reduction, significant reduction, near totalreduction, and total reduction.

“Animal protein free” means the absence of blood derived, blood pooledand other animal derived products or compounds. “Animal” means a mammal(such as a human), bird, reptile, fish, insect, spider or other animalspecies. “Animal” excludes microorganisms, such as bacteria. Thus, ananimal protein free pharmaceutical composition can include a botulinumneurotoxin. For example, an “animal protein free” pharmaceuticalcomposition means a pharmaceutical composition which is eithersubstantially free or essentially free or entirely free of a serumderived albumin, gelatin and other animal derived proteins, such asimmunoglobulins. An example of an animal protein free pharmaceuticalcomposition is a pharmaceutical composition which comprises or whichconsists of a botulinum toxin (as the active ingredient) and a suitablepolysaccharide as a stabilizer or excipient.

“Antioxidant” refers to any compound which protects an active ingredientfrom reaction with oxygen. Antioxidants can be broadly divided intothree categories: (i) sacrificial antioxidants, which react with oxygenmore readily than a particular active ingredient and therefore canscavenge oxygen, e.g., ascorbic acid and sulfites; (ii) chainterminators, which are molecules that form stable radicals due to weakbonds to hydrogen atoms that are attacked in a propagation of radicalchains by consumption of oxygen, e.g., methionine, NAC, glutathionine,lipoic acid, butylated hydroxytoluene (BHT), and cysteine, (iii)chelating agents, which reduce catalytic activity of transition metalsby forming complexes with the metals, e.g., EDTA, EGTA and DTPA andanalogs thereof.

“Biological activity” describes the beneficial or adverse effects of adrug on living matter. When a drug is a complex chemical mixture, thisactivity is exerted by the substance's active ingredient but can bemodified by the other constituents. Biological activity can be assessedas potency or as toxicity by an in vivo LD₅₀ or ED₅₀ assay, or throughan in vitro assay such as, for example, cell-based potency assays asdescribed in U.S. 2010/0203559 and U.S. 2010/0233802.

“Botulinum toxin” means a neurotoxin produced by Clostridium botulinum,as well as a botulinum toxin (or the light chain or the heavy chainthereof) made recombinantly by a non-Clostridial species. The phrase“botulinum toxin”, as used herein, encompasses the botulinum toxinserotypes A, B, C, D, E, F and G, and their subtypes and any other typesof subtypes thereof, or any re-engineered proteins, analogs,derivatives, homologs, parts, sub-parts, variants, or versions, in eachcase, of any of the foregoing. “Botulinum toxin”, as used herein, alsoencompasses a “modified botulinum toxin”. Further “botulinum toxin” asused herein also encompasses a botulinum toxin complex, (for example,the 300, 600 and 900 kDa complexes), as well as the neurotoxic componentof the botulinum toxin (150 kDa) that is unassociated with the complexproteins.

“Clostridial toxin” refers to any toxin produced by a Clostridial toxinstrain that can execute the overall cellular mechanism whereby aClostridial toxin intoxicates a cell and encompasses the binding of aClostridial toxin to a low or high affinity Clostridial toxin receptor,the internalization of the toxin/receptor complex, the translocation ofthe Clostridial toxin light chain into the cytoplasm and the enzymaticmodification of a Clostridial toxin substrate. Non-limiting examples ofClostridial toxins include Botulinum toxins, such as a BoNT/A, a BoNT/B,a BoNT/C₁, a BoNT/D, a BoNT/E, a BoNT/F, a BoNT/G, a Tetanus toxin(TeNT), a Baratii toxin (BaNT), and a Butyricum toxin (BuNT). TheBoNT/C2 cytotoxin and BoNT/C3 cytotoxin, not being neurotoxins, areexcluded from the term “Clostridial toxin.” The term Clostridial toxinalso includes the approximately 150-kDa Clostridial toxin alone (i.e.without the NAPs). A Clostridial toxin includes naturally occurringClostridial toxin variants, such as, e.g., Clostridial toxin isoformsand Clostridial toxin subtypes; non-naturally occurring Clostridialtoxin variants, such as, e.g., conservative Clostridial toxin variants,non-conservative Clostridial toxin variants, Clostridial toxin chimericvariants and active Clostridial toxin fragments thereof, or anycombination thereof. A Clostridial toxin also includes Clostridial toxincomplexes, which refers to a complex comprising a Clostridial toxin andnon-toxin associated proteins (NAPs), such as, e.g., a Botulinum toxincomplex, a Tetanus toxin complex, a Baratii toxin complex, and aButyricum toxin complex. Non-limiting examples of Clostridial toxincomplexes include those produced by a Clostridium botulinum, such as,e.g., a 900-kDa BoNT/A complex, a 500-kDa BoNT/A complex, a 300-kDaBoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDa BoNT/C₁ complex, a500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a 300-kDa BoNT/Ecomplex, and a 300-kDa BoNT/F complex.

“Clostridial toxin active ingredient” refers to a molecule whichcontains any part of a Clostridial toxin that exerts an effect upon orafter administration to a subject or patient. As used herein, the term“Clostridial toxin” encompasses (i) a Clostridial toxin complexcomprising the approximately 150-kDa Clostridial toxin and otherproteins collectively called non-toxin associated proteins (NAPs), (ii)the approximately 150-kDa Clostridial toxin alone (i.e. without theNAPs), or (iii) a modified Clostridial toxin, such as, e.g., are-targeted Clostridial toxins.

“Deformity” means a cosmetic, physical or functional irregularity,defect, abnormality, imperfection, malformation, depression, ordistortion.

“Effective amount” as applied to the biologically active ingredientmeans that amount of the ingredient which is generally sufficient toeffect a desired change in the subject. For example, where the desiredeffect is a reduction in an autoimmune disorder symptom, an effectiveamount of the ingredient is that amount which causes at least asubstantial reduction of the autoimmune disorder symptom, and withoutresulting in significant toxicity.

“Effective amount” when used in reference to the amount of an excipientor specific combination of excipients added to a Clostridial toxincomposition, refers to the amount of each excipient that is necessary toachieve the desired initial recovered potency of a Clostridial toxinactive ingredient. In aspects of this embodiment, an effective amount ofan excipient or combination of excipients results in an initialrecovered potency of, e.g., at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 100%. In other aspects of this embodiment, atherapeutically effective concentration of a Clostridial toxin activeingredient reduces a symptom associated with the aliment being treatedby, e.g., at most 10%, at most 20%, at most 30%, at most 40%, at most50%, at most 60%, at most 70%, at most 80%, at most 90% or at most 100%.

“Heavy chain” means the heavy chain of a botulinum neurotoxin. It has amolecular weight of about 100 kDa and can be referred to as the H chain,or as H.

H_(C) means a fragment (about 50 kDa) derived from the H chain of abotulinum neurotoxin which is approximately equivalent to the carboxylend segment of the H chain, or the portion corresponding to thatfragment in the intact H chain. It is believed to be immunogenic and tocontain the portion of the natural or wild type botulinum neurotoxininvolved in high affinity, presynaptic binding to motor neurons.

H_(N) means a fragment (about 50 kDa) derived from the H chain of abotulinum neurotoxin which is approximately equivalent to the amino endsegment of the H chain, or the portion corresponding to that fragment inthe intact in the H chain. It is believed to contain the portion of thenatural or wild type botulinum neurotoxin involved in the translocationof the L chain across an intracellular endosomal membrane.

“Light chain” means the light chain of a Clostridial neurotoxin. It hasa molecular weight of about 50 kDa, and can be referred to as the Lchain, L, or as the proteolytic domain (amino acid sequence) of abotulinum neurotoxin.

LH_(N) or L-H_(N) means a fragment derived from a Clostridial neurotoxinthat contains the L chain, or a functional fragment thereof coupled tothe H_(N) domain It can be obtained from the intact Clostridialneurotoxin by proteolysis, so as to remove or to modify the Hc domain.

“Implant” means a controlled release (e.g., pulsatile or continuous)composition or drug delivery system. The implant can be, for example,injected, inserted or implanted into a human body.

“Liquid composition”, “liquid pharmaceutical composition”, or “liquidformulation” refers to a pharmaceutically active preparation of drug orbiological which is capable of being stored in a liquid pharmaceuticalexcipient, such as a buffering agent, for an extended period of time,such that it can be ready-to-use as needed by a clinician. The liquidpharmaceutical composition is manufactured without a lyophilizationprocess.

“Local administration” means direct administration of a pharmaceuticalat or to the vicinity of a site on or within an animal body, at whichsite a biological effect of the pharmaceutical is desired, such as via,for example, intramuscular or intra- or subdermal injection or topicaladministration. Local administration excludes systemic routes ofadministration, such as intravenous or oral administration. Topicaladministration is a type of local administration in which apharmaceutical agent is applied to a patient's skin.

“Lyoprotector” or “lyoprotectant” means a substance that is included ina lyophilized formulation to protect a Clostridial toxin activeingredient during the freeze-drying process. Lyoprotectors include forexample polyhydroxy compounds such as sugars (mono-, di-, andpolysaccharides), polyalcohols, and their derivatives. Exemplarylyoprotectors which can be used with the lyophilized formulationsdisclosed herein include sucrose, trehalose, mannitol, sorbitol,glucose, raffinose, maltose, glycerol, lactose, fructose, galactose, andcombinations thereof.

“Lyophilized composition”, “lyophilized pharmaceutical composition”,“lyophilized formulation”, or “solid composition” refers to aformulation containing a Clostridial toxin active ingredient which hasbeen subjected to a lyophilization, freeze-drying or vacuum-dryingprocess; and can be reconstituted with a reconstitution vehicle, such asfor example saline or water, prior to administration to a patient. Thelyophilized composition can be a freeze-dried composition or avacuum-dried composition.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Mutation” means a structural modification of a naturally occurringprotein or nucleic acid sequence. For example, in the case of nucleicacid mutations, a mutation can be a deletion, addition or substitutionof one or more nucleotides in the DNA sequence. In the case of a proteinsequence mutation, the mutation can be a deletion, addition orsubstitution of one or more amino acids in a protein sequence. Forexample, a specific amino acid comprising a protein sequence can besubstituted for another amino acid, for example, an amino acid selectedfrom a group which includes the amino acids alanine, asparagine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, tyrosine or any othernatural or non-naturally occurring amino acid or chemically modifiedamino acids. Mutations to a protein sequence can be the result ofmutations to DNA sequences that when transcribed, and the resulting mRNAtranslated, produce the mutated protein sequence. Mutations to a proteinsequence can also be created by fusing a peptide sequence containing thedesired mutation to a desired protein sequence.

“Patient” means a human or non-human subject receiving medical orveterinary care. Accordingly, the compositions as disclosed herein canbe used in treating any animal, such as, for example, mammals, or thelike.

“Peripherally administering” or “peripheral administration” meanssubdermal, intradermal, transdermal, or subcutaneous administration, butexcludes intramuscular administration. “Peripheral” means in a subdermallocation, and excludes visceral sites.

“Pharmaceutical composition” means a composition comprising an activepharmaceutical ingredient, such as, for example, a Clostridial toxinactive ingredient such as a botulinum toxin, and at least one additionalingredient, such as, for example, a stabilizer or excipient or the like.A pharmaceutical composition is therefore a formulation which issuitable for diagnostic or therapeutic administration to a subject, suchas a human patient. The pharmaceutical composition can be, for example,in a lyophilized or vacuum dried condition, a solution formed afterreconstitution of the lyophilized or vacuum dried pharmaceuticalcomposition, or as a solution or solid which does not requirereconstitution.

“Pharmacologically acceptable excipient” is synonymous with“pharmacological excipient” or “excipient” and refers to any excipientthat has substantially no long term or permanent detrimental effect whenadministered to mammal and encompasses compounds such as, e.g.,stabilizing agent, a bulking agent, a cryo-protectant, a lyoprotectant,an additive, a vehicle, a carrier, a diluent, or an auxiliary. Anexcipient generally is mixed with an active ingredient, or permitted todilute or enclose the active ingredient and can be a solid, semi-solid,or liquid agent. It is also envisioned that a pharmaceutical compositioncomprising a Clostridial toxin active ingredient can include one or morepharmaceutically acceptable excipients that facilitate processing of anactive ingredient into pharmaceutically acceptable compositions. Insofaras any pharmacologically acceptable excipient is not incompatible withthe Clostridial toxin active ingredient, its use in pharmaceuticallyacceptable compositions is contemplated. Non-limiting examples ofpharmacologically acceptable excipients can be found in, e.g.,Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Anselet al., eds., Lippincott Williams & Wilkins Publishers, 7^(th) ed.1999); Remington: The Science and Practice of Pharmacy (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20^(th) ed. 2000); Goodman& Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook ofPharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003), each of which is hereby incorporated by referencein its entirety.

The constituent ingredients of a pharmaceutical composition can beincluded in a single composition (that is, all the constituentingredients, except for any required reconstitution fluid, are presentat the time of initial compounding of the pharmaceutical composition) oras a two-component system, for example a vacuum-dried compositionreconstituted with a reconstitution vehicle which can, for example,contain an ingredient not present in the initial compounding of thepharmaceutical composition. A two-component system can provide severalbenefits, including that of allowing incorporation of ingredients whichare not sufficiently compatible for long-term shelf storage with thefirst component of the two-component system. For example, thereconstitution vehicle may include a preservative which providessufficient protection against microbial growth for the use period, forexample one-week of refrigerated storage, but is not present during thetwo-year freezer storage period during which time it might degrade thetoxin. Other ingredients, which may not be compatible with a botulinumtoxin or other ingredients for long periods of time, can be incorporatedin this manner; that is, added in a second vehicle (e.g. in thereconstitution vehicle) at the approximate time of use. A pharmaceuticalcomposition can also include preservative agents such as benzyl alcohol,benzoic acid, phenol, parabens and sorbic acid. Pharmaceuticalcompositions can include, for example, excipients, such as surfaceactive agents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents;preservatives; physiologically degradable compositions such as gelatin;aqueous vehicles and solvents; oily vehicles and solvents; suspendingagents; dispersing or wetting agents; emulsifying agents, demulcents;buffers; salts; thickening agents; fillers; antioxidants; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials and other ingredients known in the art and described, forexample in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., which is incorporated herein by reference.

“Polysaccharide” means a polymer of more than two saccharide moleculemonomers. The monomers can be identical or different.

“Stabilizing agent”, “stabilization agent” or “stabilizer” means asubstance that acts to stabilize a Clostridial toxin active ingredientsuch that the potency of the pharmaceutical composition is increasedrelative to an unstabilized composition.

“Stabilizers” can include excipients, and can include protein andnon-protein molecules.

“Surfactant” refers to a natural or synthetic amphiphilic compound. Asurfactant can be non-ionic, zwitterionic, or ionic. Non-limitingexamples of surfactants include a poloxamer, a polysorbate, andcombinations thereof.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease, such as, for example, adisorder or a disease characterized by hyperactivity (i.e. spasticity)of a peripheral muscle.

Therapeutically effective concentration”, “therapeutically effectiveamount,” “effective amount,” “effective dose,” and “therapeuticallyeffective dose” refer to the minimum dose of a Clostridial toxin activeingredient necessary to achieve the desired therapeutic effect andincludes a dose sufficient to reduce a symptom associated with alimentbeing treated.

“TEM” as used herein, is synonymous with “Targeted Exocytosis Modulator”or “retargeted endopeptidase.” Generally, a TEM comprises an enzymaticdomain from a Clostridial toxin light chain, a translocation domain froma Clostridial toxin heavy chain, and a targeting domain. The targetingdomain of a TEM provides an altered cell targeting capability thattargets the molecule to a receptor other than the native Clostridialtoxin receptor utilized by a naturally-occurring Clostridial toxin. Thisre-targeted capability is achieved by replacing the naturally-occurringbinding domain of a Clostridial toxin with a targeting domain having abinding activity for a non-Clostridial toxin receptor. Although bindingto a non-Clostridial toxin receptor, a TEM undergoes all the other stepsof the intoxication process including internalization of theTEM/receptor complex into the cytoplasm, formation of the pore in thevesicle membrane and di-chain molecule, translocation of the enzymaticdomain into the cytoplasm, and exerting a proteolytic effect on acomponent of the SNARE complex of the target cell.

“Tonicity agent” means a low molecular weight excipient which isincluded in a formulation to provide isotonicity. Non-limiting examplesof a tonicity agent include a disaccharide such as trehalose or sucrose;a polyalcohol such as sorbitol or mannitol; a monosaccharide such asglucose; and a salt such as sodium chloride.

“Topical administration” excludes systemic administration of theneurotoxin. In other words, and unlike conventional therapeutictransdermal methods, topical administration of botulinum toxin does notresult in significant amounts, such as the majority of, the neurotoxinpassing into the circulatory system of the patient.

“Treating” means to alleviate (or to eliminate) at least one symptom ofa condition or disorder, such as, for example, wrinkles, spasticity,depression, pain (such as, for example, headache pain), bladderoveractivity, or the like, either temporarily or permanently.

As used herein, the term “unit” or “U” refers to the LD₅₀ dose or thedose determined by a cell based potency assay (CBPA). The LD₅₀ dose isdefined as the amount of a Clostridial toxin active ingredient,Clostridial toxin complex or modified Clostridial toxin that killed 50%of the mice injected with the Clostridial toxin, Clostridial toxincomplex or modified Clostridial toxin. The CBPA dose is determined asdescribed in U.S. Pat. No. 8,618,261, the assay details of which areincorporated by reference herein.

“Variant” means a Clostridial neurotoxin, such as wild-type botulinumtoxin serotype A, B, C, D, E, F or G, that has been modified by thereplacement, modification, addition or deletion of at least one aminoacid relative to wild-type botulinum toxin, which is recognized by atarget cell, internalized by the target cell, and catalytically cleavesa SNARE (SNAP (Soluble NSF Attachment Protein) Receptor) protein in thetarget cell.

An example of a variant neurotoxin component can comprise a variantlight chain of a botulinum toxin having one or more amino acidssubstituted, modified, deleted and/or added. This variant light chainmay have the same or better ability to prevent exocytosis, for example,the release of neurotransmitter vesicles. Additionally, the biologicaleffect of a variant may be decreased compared to the parent chemicalentity. For example, a variant light chain of a botulinum toxin type Ahaving an amino acid sequence removed may have a shorter biologicalpersistence than that of the parent (or native) botulinum toxin type Alight chain.

Pharmaceutical Compositions

In a first aspect, a pharmaceutical composition comprising (orconsisting of, or consisting essentially of) a Clostridial toxin activeingredient, a disaccharide, a surfactant and an antioxidant isdescribed. The composition can, in one embodiment, be a solidcomposition, such as a lyophilized powder that is reconstituted prior touse. In another embodiment, the composition is a liquid composition;that is, the composition is manufactured and stored in liquid form.Studies were conducted demonstrating that the compositions stabilize theClostridial toxin active ingredient to retain its potency, as will nowbe described with respect to Examples 1-11.

In one study, described in Example 1, compositions were prepared thatcomprised botulinum toxin as a model Clostridial toxin activeingredient, a disaccharide, a surfactant, and an antioxidant. Theformulations were lyophilized and stored under various temperatures forselected periods of time, ranging from 1 month to 7.5 months. Potency ofthe lyophilized formulations after storage and reconstitution withsaline was tested by a cell based potency assay. The results werenormalized to target potency. The potencies of the solid compositionsare shown in Tables 1.1, 1.2, 2 and 3.

TABLE 1.1 Lyophilized formulations Excipient, % w/w Normalized potency,TWEEN ® storage at 25° C. Formulation Treh 20 P188 NaCl Met NAC BufferT0 3 mo 6 mo Comparator 1 3 0.04 0.9 0.2 — water 80.6% 86.4% 80.8%Comparator 2 2 — 4 — — — 20 mM 81.5% 68.6% 68.0% Histidine pH 5 5Formulation 1 2 — 4 — — 0.03 20 mM 98.6% 91.9% 86.9% Histidine pH 5.5Treh = trehalose; P188 = poloxamer P188; Met = L-methionine; NAC =N-acetyl-L-cysteine.

TABLE 1.2 Lyophilized formulations Excipient, % w/w Normalized potency,TWEEN ® storage at 40° C. Formulation Treh 20 P188 NaCl Met NAC BufferT0 3 mo 6 mo Comparator 1 3 0.04 0.9 0.2 Water 74.8% 70.3% 24.0%Comparator 2 2 4 20 mM 64.8% 57.8% 46.8% Histidine pH 5.5 Formulation 12 4 0.03 20 mM 86.5% 77.9% 62.3% Histidine pH 5.5 Treh = trehalose; P188= poloxamer P188; Met = L-methionine; NAC = N-acetyl-L-cysteine.

The data in Table 1.1 demonstrates that a lyophilized botulinum toxincomposition comprised of trehalose, poloxamer, and NAC retained morethan 85% potency after storage for six months at 25° C. The formulationreferred to as Comparator 2, which was identical to Formulation 1 exceptthat it lacked an antioxidant (i.e., no methionine, no NAC), had only68% retained potency after storage for six months at 25° C. Formulation1 had about 7.5% higher potency than Comparator 1 after storage for sixmonths at 25° C. (calculated as a percent difference,((86.9%-80.8%)/80.8%)). Formulation 1 had 27.8% higher potency thanComparator 2 after storage for six months at 25° C. (calculated as apercent difference, ((86.9%-68%)/68%)). The data in Table 1.2 showspotency data for the same three formulations as Table 1.1 (Formulation 1and Comparator 1 and Comparator 2), demonstrating that a lyophilizedbotulinum toxin composition comprised of trehalose, poloxamer, and NACretained more than 60% potency after storage for six months at 40° C.The formulation referred to as Comparator 2, which was identical toFormulation 1 except that it lacked an antioxidant (e.g., no methionine,no NAC), had only 46.8% retained potency after storage for six months at40° C. Formulation 1 had about 160% higher potency than Comparator 1after storage for six months at 40° C. (calculated as a percentdifference). Formulation 1 had about 33% higher potency than Comparator2 after storage for six months at 40° C. (calculated as a percentdifference). Accordingly, in one embodiment, a lyophilized compositionis contemplated, where the composition is comprised of a Clostridialtoxin active ingredient, such as a botulinum toxin; a disaccharide; asurfactant; and an antioxidant. In one embodiment, the compositionretains potency after storage for six months at 25° C. that is at leastabout 15%, 20%, or 25% greater than a formulation lacking theantioxidant. In another embodiment, a lyophilized composition iscontemplated, where the composition is comprised of a Clostridial toxinactive ingredient, such as a botulinum toxin; a disaccharide; asurfactant; and an antioxidant. In one embodiment, the compositionretains potency after storage for six months at 40° C. that is at leastabout 20%, 25%, or 30% greater than a formulation lacking theantioxidant. In another embodiment, a lyophilized composition iscontemplated, where the composition is comprised of a Clostridial toxinactive ingredient, such as a botulinum toxin; a disaccharide; asurfactant; and an antioxidant. In one embodiment, the compositionretains at least about 55% or 60% potency after storage for six monthsat 40° C., where potency is measured in a cell-based potency assay.

It will be appreciated that potency of the Clostridial toxin can beascertained in various ways, such as the in vitro cell-based potencyassay used in the studies discussed herein or in an in vivo assay. Anexemplary in vivo assay is the LD₅₀ potency assay, where the potency ofthe toxin is expressed as a multiple of the LD₅₀ value for a mouse, oneunit (U) of toxin being defined as being the equivalent amount of toxinthat kills 50% of a group of 18 to 20 female Swiss-Webster mice,weighing about 20 grams each. Any known method for assessing potency canbe used to determine whether a composition or a particular compositioncomponents stabilize a toxin. The in vitro cell-based potency assay usedherein is not intended to be limiting to the possible approaches toascertain potency.

In another study, with continuing reference to Example 1, lyophilizedbotulinum toxin compositions were prepared with trehalose; a polysorbateor a poloxamer; and methionine or NAC. Comparator formulations wereprepared with sucrose (Comparator 3) and with no antioxidant (Comparator2). The lyophilized formulations and lyophilized comparator formulationswere placed in storage for 1 month at −20° C. or at 40° C. Potency ofthe botulinum toxin was measured using a cell-based potency assay andthe results are shown in Table 2.

TABLE 2 Lyophilized formulations Normalized potency Excipient, % w/w (%of target) TWEEN ® 1 mo 1 mo Formulation Treh Sucr 20 P188 NaCl Met NACBuffer T0 −20° C. 40° C. Comparator 2 4 20 mM  87.15%  88.95% 76.32% 2Histidine pH 5.5 Comparator 3 0.04 0.9 0.2 Water  84.06%  85.18% 72.86%3 Formulation 2 4 0.2 20 mM  98.60% 120.85% 91.17% 2 Histidine pH 6.0Formulation 8 0.04 0.03 20 mM 105.2% 110.05% 96.89% 3 Histidine pH 6.0Treh = trehalose; Sucr = sucrose; P188 = poloxamer P188; Met =L-methionine; NAC = N-acetyl-L-cysteine.

Inspection of the data in Table 2 for compositions identified asFormulation 2 and Comparator 2 demonstrates the stabilizing effect onthe botulinum toxin by the combination of a disaccharide, a surfactant,and an antioxidant. Formulation 2 and Comparator 2 were identical in allrespects other than Comparator 2 lacked an antioxidant. The potency datafor these two compositions shows that the antioxidant acts to stabilizethe toxin, as Formulation 2 retained more than 90% potency after storagefor one month at 40° C. whereas the comparative formulation (Comparator2) lacking the antioxidant had only 76% potency after storage for onemonth at 40° C. Formulation 3, comprised of trehalose, polysorbatesurfactant (TWEEN®-20), and an antioxidant NAC, retained more than 95%potency after storage for one month at 40° C., considerably better thaneither comparative formulation. Accordingly, in one embodiment, alyophilized composition is contemplated, where the composition iscomprised of a Clostridial toxin active ingredient, such as a botulinumtoxin; a disaccharide; a surfactant; and an antioxidant. In oneembodiment, the composition retains at least about 80%, 85% or 90%potency after storage for one month at 40° C., where potency is measuredin a cell-based potency assay. In another embodiment, a lyophilizedcomposition is contemplated, where the composition is comprised of aClostridial toxin active ingredient, such as a botulinum toxin; adisaccharide; a surfactant selected from a poloxamer and a polysorbate;and an antioxidant selected from methionine, NAC, EDTA, EGTA, DTPA,analogs thereof, and combinations thereof. In one embodiment, thecomposition retains at least about 80%, 85% or 90% potency after storagefor one month at 40° C., where potency is measured in a cell-basedpotency assay. Such compositions, in another embodiment, retain potencyafter storage for one month at 40° C. that is at least about 18%, 20% or24% greater than a formulation lacking the antioxidant.

With continued reference to Example 1, compositions comprised of aClostridial toxin active ingredient (botulinum toxin), trehalose, apoloxamer surfactant (e.g. KOLLIPHOR® P-188) and methionine as astabilizing antioxidant were prepared. A comparative formulation lackingthe methionine was prepared (Comparator 2). The compositions werelyophilized and placed in storage for 7.5 months at 25° C. Potency ofthe botulinum toxin was measured after 3 months and after 7.5 months ofstorage using a cell-based potency assay and normalized to the targetpotency. The results are shown in Table 3.

TABLE 3 Lyophilized formulations Normalized potency, Excipient, % w/wstorage at 25° C. Formulation Trehalose P-188 Met Buffer T0 3 mo 7.5 moComparator 2 2 4 20 mM 87.1% 78.0% 78.0% Histidine, pH 5.5 Formulation 22 4 0.2 20 mM 98.6% 97.0% 98.0% Histidine, pH 6.0 Formulation 4 8 0.60.2 20 mM 86.5% 83.0% 84.0% Histidine, pH 6.0 P-188 = Poloxamer P-188;Met = L-methionine.

Inspection of the data in Table 3 for compositions identified asFormulation 2 and Comparator 2 demonstrates the stabilizing effect onthe botulinum toxin by the combination of a tonicity agent, asurfactant, and an antioxidant. Formulation 2 and Comparator 2 wereidentical in all respects other than Comparator 2 lacked the antioxidantand had a slightly lower pH. The potency data for these two compositionsshows that the antioxidant acts to stabilize the toxin, as Formulation 2retained more than 95% potency after storage for 3 months and afterstorage for 7.5 months at 25° C. whereas the comparative formulationlacking the antioxidant had only 78% potency after storage for 3 monthsand after storage for 7.5 months at 25° C. Formulation 4 was comprisedof the same components as Formulation 2, but with a higher weightpercent of trehalose and a lower weight percent of poloxamer surfactant.Formulation 4 also stabilized the botulinum toxin better than theComparator 2 formulation that lacked methionine. Accordingly, in oneembodiment, a lyophilized composition is contemplated, where thecomposition is comprised of a Clostridial toxin active ingredient, suchas a botulinum toxin; trehalose; a poloxamer surfactant; and methionine.In one embodiment, the composition retains at least about 80%, 85% or90% potency after storage for three months at 25° C., where potency ismeasured in a cell-based potency assay. In another embodiment, alyophilized composition is contemplated, where the composition iscomprised of a Clostridial toxin active ingredient, such as a botulinumtoxin; trehalose; a poloxamer surfactant; and methionine. In oneembodiment, the composition retains potency after storage for threemonths at 25° C. that is at least about 10%, 15%, or 20% greater than aformulation lacking the antioxidant.

Example 2 describes another study where liquid compositions comprised ofa Clostridial toxin active ingredient, a tonicity agent, a surfactant,and an antioxidant were prepared. Liquid solutions of a Clostridialtoxin active ingredient, using botulinum toxin as a model, were preparedwith a disaccharide tonicity agent, a poloxamer surfactant, and anantioxidant were prepared. In this particular study, the disaccharidetonicity agent was trehalose and the poloxamer surfactant was poloxamerP188. Three formulations were prepared, each with the same amount ofbotulinum toxin, 8 w/w % trehalose, and 4 w/w % poloxamer P188 inhistidine buffer. The target potency was 100 Units/ml. Formulation 10had no antioxidant; Formulation 11 contained NAC, and Formulation 12contained L-methionine. The liquid compositions were placed in glassvials and stored at −70° C., 5° C., 25° C. and 40° C. for one month.Potency of the botulinum toxin was measured prior to storage and afterone month of storage using a cell-based potency assay. Results are shownin Table 4.

TABLE 4 Liquid formulations Potency, U/mL Formulation 1 mo. 1 mo. 1 mo.1 mo. No.* Antioxidant T0 −70° C. 5° C. 25° C. 40° C. Formulation None128 135 135 106 0.225 10 Formulation N-acetyl-L- 128 133 129 61 0.2 11cysteine, 0.2 % w/w Formulation L-methionine, 133 146 146 145 138 120.2% w/w *Each formulation contained the same amount of botulinum toxin,8 w/w % trehalose and 4 w/w % poloxamer P188 in histidine buffer.

The liquid composition comprising methionine (Formulation 12) retainedits potency after one month storage at all four temperatures, including40° C., whereas the liquid composition with no antioxidant (Formulation10) lost approximately 17% potency after storage for 1 month at 25° C.and lost essentially all activity (i.e., complete inactivation of thetoxin) after storage for 1 month at 40° C. The liquid compositioncomprising NAC lost over 50% potency after storage for 1 month at 25° C.and lost essentially all activity (i.e., complete inactivation of thetoxin) after storage for 1 month at 40° C., suggesting thatN-acetyl-L-cysteine can act as a pro-oxidant in this formulation. Incontrast, as shown in Tables 1.1 and 1.2, the lyophilized compositionscomprising NAC lost 7% ((98.6-91.9)/98.6) potency after storage for 3months at 25° C. and lost 12% ((98.6-86.5)/98.6) potency after storagefor 1 month at 40° C. This demonstrates that NAC by itself can functionas a stabilizer in the lyophilized compositions.

Further studies were conducted to investigate the impact of antioxidantcompounds on the liquid compositions. In Example 3, liquid compositionswere prepared with 100 U/mL botulinum toxin, 8 w/w % trehalose, and 4w/w % poloxamer P188 in histidine buffer at pH 6.0. Each formulation hada different antioxidant or a combination of antioxidants, as set forthin Table 5 below and in Example 3. The antioxidants tested included NAC,L-methionine, L-tryptophan, L-glutathione, sodium sulfite, propylgallate, and EDTA sodium salt. Potency of the formulations was tested bya cell based potency assay after filling (time zero, t0) and afterstorage at 40° C. for 2 weeks and 1 month. Potency test results areshown in Table 5.

TABLE 5 Liquid formulations Antioxidant² Potency U/mL Formulation NACMet TRP GSH NaSul PrpGal EDTA 2 wks 1 mo. No.¹ % % % % % % % T0 40° C.40° C. Formulation 0.2 126 129 130 20 Formulation 0.2 127 13.71 NT³ 21Formulation 0.2 123 3.76 NT 22 Formulation 0.2 23.7 0.161 NT 23Formulation 0.2 0.164 0.150 NT 24 Formulation 0.2 0.2 133 0.253 NT 25Formulation 0.2 0.03 129 127 127 26 Formulation 0.2 0.2 0.03 129 125 12227 Formulation 0.2 0.2 126 2.45 NT 28 ¹Each formulation contained 100U/mL botulinum toxin, 8 w/w % trehalose, and 4 w/w % poloxamer P188 in20 mM histidine buffer, pH 6.0 and the specified antioxidant. ²NAC =N-acetyl-L-cysteine; Met = L-methionine; TRP = L-tryptophan; GSH =L-glutathione; NaSul = sodium sulfite; PrpGal = propyl gallate; EDTA =ethylene diamine tetraacetic acid, sodium salt. ³NT = not tested

The stabilizing effect of the antioxidant, or lack of stabilizing effectfor certain antioxidants, is apparent from this study. Compositionscomprising methionine (Formulation 20) retained full potency afterstorage at 40° C. for 2 weeks and 1 month, in contrast to compositionscomprising tryptophan (Formulation 21) or glutathione (Formulation 22),indicating that not all amino acids function to stabilize a Clostridialtoxin active ingredient in a liquid composition. Compositions comprisingsodium sulfite (Formulation 23) or propyl gallate (Formulation 24) lostessentially all potency after 2 weeks storage at 40° C. Liquidcompositions comprising NAC and EDTA sodium salt (Formulation 26)retained full potency after storage at 40° C. for 2 weeks and 1 month,as did a composition comprising NAC, EDTA sodium salt and tryptophan(Formulation 27). In contrast, as shown in Table 4, liquid compositionscomprising NAC but not EDTA (Formulation 25) lost essentially allpotency after 2 weeks storage at 40° C. This demonstrates that thecombination of antioxidants—a chelating agent (e.g., EDTA, EGTA, DTPA oranalogs thereof) and/or a chain terminator antioxidant (e.g.,methionine, cysteine, NAC or BHT) provides a stabilizing effect on thebotulinum toxin. Accordingly, compositions in liquid or solution formcomprising a Clostridial toxin active ingredient; a disaccharide; asurfactant; and an antioxidant selected from methionine, NAC, EDTA,EGTA, DTPA, analogs thereof, and combinations thereof, are contemplated.In one embodiment, the composition retains potency of the toxin for aperiod of at least about 2 weeks when stored at about 40° C., where thepotency is measured in a cell-based potency assay. In anotherembodiment, the composition retains potency of the toxin for a period ofat least about 1 month when stored at about 40° C., where the potency ismeasured in a cell-based potency assay. In a further embodiment, thecomposition is in liquid or solution form comprising a Clostridial toxinactive ingredient; a disaccharide; a surfactant; and an antioxidantselected from (i) methionine, and (ii) NAC and a chelating agentselected from EDTA, EGTA, DTPA, and analogs thereof. In one embodiment,the composition retains potency of the toxin for a period of at leastabout 1 month when stored at about 40° C., where the potency is measuredin a cell-based potency assay.

In another embodiment, a liquid composition comprises a Clostridialtoxin active ingredient; a tonicity agent; a surfactant; and anantioxidant selected from the group consisting of a sacrificialantioxidant, a chelating agent antioxidant, a chain terminatorantioxidant, and combinations thereof.

Example 4 details another study designed to investigate the effect of atonicity agent on stability of the toxin in the composition. In thisstudy liquid compositions were prepared with 100 U/mL botulinum toxin, 4w/w % poloxamer P188, 0.2 w/w % methionine, and either 8 w/w % trehaloseor sucrose, in histidine buffer at pH 6.0. The two compositions werestored at 25° C. and potency was tested by a cell based potency assayprior to storage (time zero, t0) and after storage. Potency test resultsare shown in Table 6.

TABLE 6 Liquid formulations Potency, U/mL Formulation at T0 and afterindicated time (in months) at 25° C. No.* Disaccharide T0 1 mo 5.5 mo 6mo 8 mo 10.5 mo Formulation 30 trehalose 133 145 — 147 159 43 (8 w/w %)Formulation 31 sucrose 117 — 10 — — — (8 w/w %) *Each formulationcontained 100 U/mL botulinum toxin, 4 w/w % poloxamer P188 and 0.2 w/w %methionine in histidine buffer.

Comparing the potency of the toxin in Formulation 30 after 6 months ofstorage at 25° C. with that in Formulation 31 after 5.5 months ofstorage at 25° C. it is apparent that trehalose affords a stabilizingeffect in a composition with poloxamer and methionine, as Formulation 30with trehalose retained 110% of its potency (147 U/mL/133 U/mL) after 6months storage at 25° C., whereas Formulation 31 with sucrose had only8.5% of the target toxin potency (10 U/mL/117 U/mL) after 5.5 monthsstorage at 25° C. Accordingly, in one embodiment, a liquid compositioncomprised of a Clostridial toxin active ingredient, such as a botulinumtoxin; trehalose; a poloxamer surfactant; and methionine iscontemplated. In one embodiment, the composition retains at least about80%, 85%, 90% or 95% potency after storage for 1, 2, 3, 4, 5, or 6months at 25° C., where potency is measured in a cell-based potencyassay. In an alternative embodiment, the composition retains at leastabout 80%, 85%, 90% or 95% potency after storage for 1, 2, 3, 4, 5, or 6months at 25° C., where potency is measured using an in vivo LD₅₀potency assay.

Further studies were conducted on liquid compositions as described inExample 5. In this study, compositions comprising a Clostridial toxinactive ingredient, poloxamer P188 (4 w/w %) or polysorbate (TWEEN® 20,0.04 w/w %), trehalose (8% w/w), and methionine (0.2 w/w %), in 20 mMhistidine buffer at pH 6.0, were prepared. The composition withpoloxamer P188 was identified as Formulation No. 30 and the compositionwith polysorbate was identified as Formulation No. 32. Potency wastested by a cell based potency assay prior to storage (time zero, t0)and after 1 month of storage 40° C. for 1 month. Potency test resultsare shown in Table 7.

TABLE 7 Liquid formulations Potency, U/mL at time zero and Formulationafter 1 month storage at 40° C. No.* Surfactant T0 1 mo. Formulationpoloxamer P188 133 138 30 (4 w/w %) Formulation polysorbate 114 9 32(TWEEN ® 20, 0.04 w/w %) *Each formulation contained 100 U/mL botulinumtoxin, 8 w/w % trehalose, and 0.2 w/w % methionine in histidine buffer.

Comparing the potency of the toxin in Formulation 30 after 1 month ofstorage at 40° C. with that in Formulation 32 it is apparent that apoloxamer surfactant affords a stabilizing effect in a composition withtrehalose and methionine, as Formulation 30 with poloxamer P188 retainedabout 104% of its initial potency (138 U/mL/133 U/mL) after one month ofstorage at 40° C., whereas Formulation 32 with polysorbate had onlyabout 8% of the initial toxin potency (9 U/mL/114 U/mL) after one monthof storage at 40° C. Accordingly, in one embodiment, a liquidcomposition comprised of a Clostridial toxin active ingredient, such asa botulinum toxin; trehalose; a poloxamer surfactant; and methionine isprovided. In one embodiment, the composition retains at least about 80%,85%, or 90% potency after storage for 1 month at 40° C., where potencyis measured in a cell-based potency assay.

In another study, five liquid compositions were prepared with botulinumtoxin as a model Clostridial toxin active ingredient. The compositionswere prepared with or without poloxamer surfactant, with or withouttrehalose, and with or without methionine. Details of the compositionsare given in Example 6. Potency of Formulations 33-37 was tested by acell based potency assay prior to storage (time zero, t0) and after 1month of storage at −70° C. and 40° C. for 1 month. Potency test resultsare shown in Table 8.

TABLE 8 Liquid formulations Composition Components* Potency U/mLFormulation Toxin Trehalose Poloxamer Methionine 1 mo. 1 mo. No. U/mL %P188 % % T0 −70° C. 40° C. Formulation 100 8 4 0 128 135 0.225 33Formulation 150 0 0 0.2 52 13 0 34 Formulation 150 8 0 0.2 131 145 30 35Formulation 100 0 4 0.2 128 142 103 36 Formulation 100 8 4 0.2 126 — 13037 *Each formulation was in 20 mM histidine buffer at pH 6.0.

The potency of the botulinum toxin in Formulation 36 and in Formulation37 after 1 month of storage at −70° C. or at 40° C. reveals that liquidcompositions with poloxamer and methionine, with or without adisaccharide such as trehalose, act to stabilize the toxin. Thisdemonstrates that poloxamer and methionine can stabilize the botulinumtoxin in liquid compositions without a disaccharide. The results fromFormulation 33 suggest that methionine is a stabilizer, as Formulation33 had little toxin potency after storage in contrast to Formulation 37and Formulation 36. The results from Formulations 34 and 35 suggest thata surfactant also has a stabilizing effect on the toxin. Accordingly, inone embodiment, a liquid composition comprised of a Clostridial toxinactive ingredient, such as a botulinum toxin; a poloxamer surfactant;and an antioxidant such as methionine is contemplated. In oneembodiment, the composition retains at least about 70%, 75%, or 85%,potency after storage for 1 month at 40° C., where potency is measuredin a cell-based potency assay. In another embodiment, a disaccharide isalso included. In one embodiment, the composition retains at least about80%, 85% or 90% potency after storage for 1 month at 40° C., wherepotency is measured in a cell-based potency assay. In an alternativeembodiment, the composition retains at least about 80%, 85% or 90%potency after storage for 1 month at 40° C., where potency is measuredusing an in vivo LD₅₀ potency assay.

In another study, described in Example 7, liquid compositions wereprepared with botulinum toxin as a model Clostridial toxin activeingredient. The compositions were prepared with a poloxamer surfactantor with a polysorbate surfactant, with or without a disaccharide. Allformulations comprised methionine. Details of the compositions are givenin Example 7. Potency of the compositions, identified as Formulations31, 38, 39 and 40, was tested by a cell based potency assay prior tostorage (time zero, t0) and after storage at 5° C. for 5.5 months, 7.5months and 12 months. Potency test results are shown in Table 9.

TABLE 9 Liquid formulations Potency U/mL Composition Components¹ afterstorage for indicated Formulation Treh Suc NaCl P188 TWEEN ®20 MetBuffer² time (in months) at 5° C. No. % % % % % % or water T0 5.5 mo 7.5mo 12 mo Formulation 8 4 0.2 buffer 117 88 NT 94 31 Formulation 8 4 0.2water 114 NT³ 108 91 38 Formulation 0.9 4 0.2 buffer  94 NT  80 62 39Formulation 8 0.04 0.2 buffer 114 NT 117 91 40 ¹All formulationscontained 100 U/mL botulinum toxin; Treh = trehalose; Suc = sucrose;P188 = poloxamer P188; TWEEN ®20 = polysorbate; Met = L-methionine;²Buffer = 20 mM His, pH 6.0; ³NT = not tested

The potency of the botulinum toxin in Formulation 31 and in Formulation38 after 12 months of storage at 5° C. reveals that the histidine bufferis not acting to stabilize the composition, as Formulation 38 wasprepared with water and was stabilized to the same extent by thesurfactant, disaccharide and methionine as Formulation 31 which wasprepared with histidine buffer. Formulation 40 prepared with trehalose,polysorbate and methionine successfully stabilized the botulinum toxinas evident from the 91 U/mL potency 12 months of storage at 5° C.Accordingly, in one embodiment, a liquid composition comprised of aClostridial toxin active ingredient, such as a botulinum toxin; apoloxamer or polysorbate surfactant; trehalose or sucrose; and anantioxidant such as methionine is contemplated. In one embodiment, thecomposition retains at least about 75%, 80%, or 85%, potency afterstorage for 1, 3, 6, 9, or 12 months at 5° C., where potency is measuredin a cell-based potency assay. In an alternative embodiment, thecomposition retains at least about 75%, 80%, or 85%, potency afterstorage for 1, 3, 6, 9, or 12 months at 5° C., where potency is measuredusing an in vivo LD₅₀ potency assay.

In another study, described in Example 8, liquid compositions with notonicity agent were prepared. Botulinum toxin was used as a modelClostridial toxin active ingredient. The compositions were prepared witheither a poloxamer surfactant or with a polysorbate surfactant, and withmethionine as the antioxidant. Details of the compositions are given inExample 8. Potency of the compositions, identified as Formulations 41and 42, was tested by a cell based potency assay prior to storage (timezero, t0) and after storage for two weeks at −70° C. and at 40° C.Potency test results are shown in Table 10.

TABLE 10 Liquid formulations Composition Components* Potency U/mL Formu-Polysorbate after storage lation Toxin P188 (TWEEN ®20) methionine for 2weeks No. U/ML % % % −70° C. 40° C. Formu- 100 0 0.04 0.2 25 1 lation 41Formu- 100 4 0 0.2 105 72 lation 42 *Both formulations were in 20 mMhistidine buffer at pH 6.0. P188 = poloxamer P188; Met = L-methionine

The potency of the botulinum toxin in Formulation 41 and in Formulation42 after 2 weeks of storage at −70° C. and at 40° C. reveals higherpotency recoveries were achieved in liquid compositions comprisingpoloxamer as the surfactant, as compared to liquid compositionscomprising polysorbate as the surfactant.

In another study, liquid compositions were prepared comprising aClostridial toxin active ingredient; poloxamer P188 (4% w/w or 0.6%w/w); trehalose (2% w/w or 8% w/w); and an antioxidant—(i) EDTA and NAC(0.03% w/w and 0.2% w/w, respectively), or (ii) methionine (0.2% w/w);in 20 mM histidine buffer at pH 6.0. Each formulation had between 30-200U botulinum toxin per vial. A summary of the compositions is set forthin Table 11.

TABLE 11 Liquid formulations Tonicity Agent Surfactant AntioxidantTrehalose Poloxamer Meth- Toxin or Sucrose NaCl P188 ionine EDTA NACU/mL wt % wt % wt % wt % wt % wt % Buffer 30-200 8 4 0.2 20 mM His, pH6.0 30-200 8 0.6 0.2 20 mM His, pH 6.0 30-200 2 0.6 4 0.2 20 mM His, pH6.0 30-200 8 4 0.03 0.2 20 mM His, pH 6.0 30-200 8 4 0.2 0.03

Accordingly, in one embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a tonicity agent selected from trehalose, sucrose, sodiumchloride, mannitol, sorbitol, glucose and combinations thereof asurfactant selected from a poloxamer, a polysorbate and combinationsthereof and an antioxidant selected from methionine, NAC, EDTA, EGTA,DTPA, analogs thereof, and combinations thereof. In one embodiment, thecomposition excludes albumin, and in another embodiment, when theantioxidant is methionine the composition excludes a polysorbate.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof and an antioxidant selected from methionine, NAC,EDTA, EGTA, DTPA, analogs thereof, and combinations thereof. In oneembodiment, the composition excludes albumin. In one embodiment, thecomposition excludes a tonicity agent.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a tonicity agent selected from trehalose, sucrose, sodiumchloride, mannitol, sorbitol, glucose, and combinations thereof; apoloxamer; and methionine. In one embodiment, the composition excludesalbumin.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a Clostridial toxin activeingredient; a disaccharide tonicity agent in an amount between 1-15 wt%; a poloxamer in an amount between 0.5-8 wt %; and an antioxidant in anamount between 0.05-5 wt %. In another embodiment, a compositioncomprises a Clostridial toxin active ingredient; trehalose in an amountbetween 1-15 wt %; a poloxamer in an amount between 0.5-8 wt %; andmethionine in an amount between 0.05-5 wt %. In one embodiment, thecomposition excludes albumin. In another embodiment, a liquidpharmaceutical composition comprises a Clostridial toxin activeingredient; trehalose in an amount between 2-15 wt % or 1-10 wt %; apoloxamer in an amount between 0.5-8 wt %; and methionine in an amountbetween 0.05-5 wt %. In one embodiment, the composition excludesalbumin.

In another embodiment, a liquid pharmaceutical composition iscontemplated. The composition comprises a botulinum toxin; a tonicityagent selected from trehalose, sucrose, sodium chloride, mannitol,sorbitol, glucose, and combinations thereof; a poloxamer; and anantioxidant selected from methionine, NAC, EDTA, EGTA, DTPA, analogsthereof, and combinations thereof. In one embodiment, the compositionexcludes albumin.

In any of the foregoing embodiments, it is contemplated that thecomposition is not, in some embodiments, an emulsion and/or excludesnanoparticles comprising an amphiphilic entity.

Lyophilized compositions were prepared for a study described in Example9. The compositions that were prepared comprised botulinum toxin as amodel Clostridial toxin; a disaccharide; a surfactant; and methionine asan antioxidant. The formulations were lyophilized and stored for twoweeks at −20° C. or at 40° C. Potency of the lyophilized formulationsafter storage and reconstitution with saline was tested by a cell basedpotency assay. The potencies of the solid compositions are shown inTable 12.

TABLE 12 Lyophilized formulations Composition Components* Potency U/mLFormulation Trehalose Sucrose TWEEN ®20 Poloxamer Methionine 2 weeks 2weeks No. % % % P188 % % −20° C. 40° C. Formulation 8 0 0 4 0.2 166 16243 Formulation 8 0 0.04 0 0.2 147 137 44 Formulation 0 8 0 4 0.2 149 13445 Formulation 0 8 0.04 0 0.2 150 142 46 *Each formulation comprised 200U/vial BoNT/A and was in 20 mM histidine buffer at pH 6.0.

A comparison of the potency data for lyophilized Formulation 43 andFormulation 45 permits analysis of the impact of trehalose versussucrose as the lyoprotector on stability of botulinum toxin inlyophilized compositions, with a poloxamer as the surfactant, andmethionine as an antioxidant. Formulations 43 and 45 differ only in thatthe former has trehalose and the latter has sucrose; both formulationscomprised a poloxamer and methionine. The potency of the botulinum toxinin Formulation 43 and in Formulation 45 after 2 weeks of storage at −20°C. and at 40° C. reveals a higher potency recovery was achieved inlyophilized compositions comprising trehalose, as compared tolyophilized compositions comprising sucrose. Accordingly, in oneembodiment, a lyophilized composition is provided, where the compositioncomprises a Clostridial toxin active ingredient, a poloxamer,methionine, and trehalose. In one embodiment, the toxin has a potencyrecovery of at least about 75% or at least about 80% after storage attwo weeks at −20° C. and/or at 40° C.

A comparison of the potency data for Formulation 43 and Formulation 44permits analysis of polysorbate and poloxamer as surfactants onstability of botulinum toxin in lyophilized compositions, with trehaloseas a lyoprotector, and methionine as an antioxidant. Formulations 43 and44 differ only in that the former has a poloxamer surfactant and thelatter has polysorbate as the surfactant. The potency of the botulinumtoxin in Formulation 43 and in Formulation 44 after 2 weeks of storageat −20° C. and at 40° C. reveals a higher potency recovery was achievedin lyophilized compositions comprising a poloxamer surfactant, ascompared to lyophilized compositions comprising a polysorbatesurfactant, when trehalose and methionine are present. Accordingly, inone embodiment, a lyophilized composition is provided, where thecomposition comprises a Clostridial toxin active ingredient, apoloxamer, methionine, and trehalose. In one embodiment, the toxin has apotency recovery of at least about 75% or at least about 80% afterstorage at two weeks at −20° C. and/or at 40° C.

Accordingly, in one embodiment, a solid or lyophilized pharmaceuticalcomposition is contemplated. The composition comprises a Clostridialtoxin active ingredient; a tonicity agent and/or lyoprotector selectedfrom trehalose, sucrose, mannitol, sorbitol, glucose, and combinationsthereof; a surfactant selected from a poloxamer, a polysorbate andcombinations thereof; and an antioxidant selected from methionine, NAC,EDTA, EGTA, DTPA, analogs thereof, and combinations thereof. In someembodiments, the solid composition comprises a lyoprotector. In someembodiments, the lyoprotector includes sucrose, trehalose, mannitol,sorbitol, glucose, or combinations thereof. In certain embodiments, thelyophilized composition is reconstituted with a tonicity agent selectedfrom trehalose, sucrose, sodium chloride, mannitol, sorbitol, glucose,and combinations thereof. In at least one embodiment, the lyophilizedcomposition is reconstituted with a reconstitution vehicle comprisingNaCl prior to administration to a patient. In at least one embodiment,NaCl is present in an amount of 0.9% (w/w) in the reconstitutionvehicle. In one embodiment, the composition excludes albumin, ahydroxyalkyl starch, glutamic acid, glutamine, aspartic acid,asparagine, a polyalcohol, glycine, and/or polyvinylpyrrolidone.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a Clostridial toxin activeingredient; trehalose or sucrose; a poloxamer; and methionine. In oneembodiment, the composition excludes albumin, a hydroxyalkyl starch,glutamic acid, glutamine, aspartic acid, asparagine, a polyalcohol,glycine, and/or polyvinylpyrrolidone. In one embodiment, the lyophilizedpharmaceutical composition comprises botulinum toxin as the Clostridialtoxin active ingredient, trehalose, a poloxamer, and methionine.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a Clostridial toxin activeingredient; trehalose in an amount between 1-15 wt %; a poloxamer in anamount between 0.5-8 wt %; and methionine in an amount between 0.05-5 wt%. In another embodiment, the composition comprises a Clostridial toxinactive ingredient, such as botulinum toxin; 8 wt % trehalose; 4 wt %poloxamer; and 0.2 wt % methionine. In one embodiment, the botulinumtoxin is present in an amount of about 200 units. In another embodiment,the botulinum toxin is present in an amount of about 50 units. In oneembodiment, the composition excludes albumin, a hydroxyalkyl starch,glutamic acid, glutamine, aspartic acid, asparagine, a polyalcohol,glycine, and/or polyvinylpyrrolidone.

In another embodiment, a solid or lyophilized pharmaceutical compositionis contemplated. The composition comprises a botulinum toxin; adisaccharide; a poloxamer; and an antioxidant selected from methionine,NAC, EDTA, EGTA, DTPA, analogs thereof, and combinations thereof. In oneembodiment, the composition excludes albumin, a hydroxyalkyl starch,glutamic acid, glutamine, aspartic acid, asparagine, a polyalcohol,glycine, and/or polyvinylpyrrolidone.

In another embodiment, a lyophilized composition comprises a Clostridialtoxin active ingredient; a tonicity agent and/or a lyoprotector; asurfactant; and an antioxidant selected from the group consisting of asacrificial antioxidant, a chelating agent antioxidant, a chainterminator antioxidant, and combinations thereof. In some embodiments,the lyophilized Clostridial pharmaceutical composition comprises alyoprotector. In some embodiments, the lyoprotector includes sucrose,trehalose, mannitol, sorbitol, glucose, or combinations thereof.

In any of the foregoing embodiments of solid or liquid compositions, itis contemplated that one or more, in any combination, of theseingredients are excluded: polyvinylpyrrolidone, diblock copolymers ofpolypropylene glycol and polyethylene glycol, and/or a polyalcohol suchas inositol, lactilol, isomalt, xylitol, or erythritol.

Pharmaceutical Composition Components

The present pharmaceutical compositions include a Clostridial toxin or aClostridial toxin active ingredient. A skilled artisan will appreciatethat the description herein refers to a Clostridial toxin activeingredient, however, a Clostridial toxin may also be used in thecompositions described herein. Accordingly, the term Clostridial toxinactive ingredient will be used; however it should be understood that aClostridial toxin is equally contemplated. In one embodiment, atherapeutically effective concentration of a Clostridial toxin activeingredient is present in the composition. In one embodiment, theClostridial toxin active ingredient reduces a symptom associated withthe aliment being treated by, e.g., at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90% or at least 100%. In other aspects of this embodiment,a therapeutically effective concentration of a Clostridial toxin activeingredient reduces a symptom associated with the aliment being treatedby, e.g., at most 10%, at most 20%, at most 30%, at most 40%, at most50%, at most 60%, at most 70%, at most 80%, at most 90% or at most 100%.

It is envisioned that any amount of Clostridial toxin active ingredientcan be added in formulating a Clostridial toxin active ingredientpharmaceutical compositions disclosed herein, with the proviso that atherapeutically effective amount of Clostridial toxin active ingredientis recoverable. In aspects of this embodiment, the amount of Clostridialtoxin active ingredient added to the formulation is at least 0.1 U/ml,at least 1.0 U/ml, at least 10 U/ml, at least 50 U/ml, at least 100U/ml, at least 200 U/ml, or at least 1000 U/ml. In other aspects of thisembodiment, the amount of Clostridial toxin active ingredient added tothe formulation is at most 0.1 U/ml, at most 1.0 U/ml, at most 10 U/ml,at most 50 U/ml, at most 100 U/ml, at most 200 U/ml, or at most 1000U/ml. In yet other aspects of this embodiment, the amount of Clostridialtoxin active ingredient added to the formulation is from about 0.1 U/mlto about 1000 U/ml, or about 1.0 U/ml to about 1000 U/ml. In still otheraspects of this embodiment, the amount of Clostridial toxin activeingredient added to the formulation is from about 0.001 U/ml to about100 U/ml, about 0.01 U/ml to about 100 U/ml, about 0.1 U/ml to about 100U/ml, or about 1.0 U/ml to about 100 U/ml.

In other aspects of this embodiment, the amount of Clostridial toxinactive ingredient added to the formulation is at least 1.0 pg, at least10 pg, at least 100 pg, at least 1.0 ng, at least 10 ng, at least 100ng, at least 1.0 μg, at least 10 μg, at least 100 μg, or at least 1.0mg. In still other aspects of this embodiment, the amount of Clostridialtoxin active ingredient added to the formulation is at most 1.0 pg, atmost 10 pg, at most 100 pg, at most 1.0 ng, at most 10 ng, at most 100ng, at most 1.0 μg, at most 10 μg, at most 100 μg, or at most 1.0 mg. Instill other aspects of this embodiment, the amount of Clostridial toxinactive ingredient added to the formulation is about 1.0 pg to about 10μg, about 10 pg to about 10 μg, about 100 pg to about 10 μg, about 1.0ng to about 10 μg, about 10 ng to about 10 μg, or about 100 ng to about10 μg. In still other aspects of this embodiment, the amount ofClostridial toxin active ingredient added to the formulation is about1.0 pg to about 1.0 μg, about 10 pg to about 1.0 μg, about 100 pg toabout 1.0 μg, about 1.0 ng to about 1.0 μg, about 10 ng to about 1.0 μg,or about 100 ng to about 1.0 μg. In further aspects of this embodiment,the amount of Clostridial toxin active ingredient added to theformulation is about 1.0 pg to about 5.0 about 10 pg to about 5.0 about100 pg to about 5.0 about 1.0 ng to about 5.0 about 10 ng to about 5.0or about 100 ng to about 5.0 μg. In further aspects of this embodiment,the amount of Clostridial toxin active ingredient added to theformulation is about 1.0 pg to about 10 about 10 pg to about 10 about100 pg to about 10 about 1.0 ng to about 10 about 10 ng to about 10 orabout 100 ng to about 10 μg.

In aspects of this embodiment, a Clostridial toxin pharmaceuticalcomposition comprises a BoNT/A, a BoNT/B, a BoNT/C₁, a BoNT/D, a BoNT/E,a BoNT/F, a BoNT/G, a mosaic BoNT such as for example BoNT/DC, a TeNT, aBaNT, or a BuNT. In another embodiment, a Clostridial toxinpharmaceutical composition comprises a Clostridial toxin variant as theClostridial toxin. In aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises naturally-occurring Clostridialtoxin active ingredient variant or a non-naturally-occurring Clostridialtoxin variant. In other aspects of this embodiment, a Clostridial toxinpharmaceutical composition comprises a BoNT/A variant, a BoNT/B variant,a BoNT/C₁ variant, a BoNT/D variant, a BoNT/E variant, a BoNT/F variant,a BoNT/G variant, a TeNT variant, a BaNT variant, or a BuNT variant,where the variant is either a naturally-occurring variant or anon-naturally-occurring variant.

Aspects of the present pharmaceutical compositions provide, in part, aClostridial toxin complex as a Clostridial toxin active ingredient. Asused herein, the term “Clostridial toxin complex” refers to a complexcomprising a Clostridial toxin and associated NAPs, such as, e.g., aBotulinum toxin complex, a Tetanus toxin complex, a Baratii toxincomplex, and a Butyricum toxin complex. Non-limiting examples ofClostridial toxin complexes include those produced by a Clostridiumbotulinum, such as, e.g., a 900-kDa BoNT/A complex, a 500-kDa BoNT/Acomplex, a 300-kDa BoNT/A complex, a 500-kDa BoNT/B complex, a 500-kDaBoNT/C₁ complex, a 500-kDa BoNT/D complex, a 300-kDa BoNT/D complex, a300-kDa BoNT/E complex, and a 300-kDa BoNT/F complex. Clostridial toxincomplexes can be purified using the methods described in Schantz, supra,(1992); Hui Xiang et al., Animal Product Free System and Process forPurifying a Botulinum Toxin, U.S. Pat. No. 7,354,740, each of which ishereby incorporated by reference in its entirety. Clostridial toxincomplexes can be obtained from, e.g., List Biological Laboratories, Inc.(Campbell, Calif.), the Centre for Applied Microbiology and Research(Porton Down, U.K), Wako (Osaka, Japan), and Sigma Chemicals (St Louis,Mo.).

Aspects of the present pharmaceutical compositions provide, in part, anon-protein excipient. As used herein, the term “non-protein excipient”refers to any excipient that is not a polypeptide comprising at leastfifteen amino acids. It is envisioned that any non-protein excipient isuseful in formulating a Clostridial toxin active ingredientpharmaceutical compositions disclosed in the present specification, withthe proviso that a therapeutically effective amount of the Clostridialtoxin active ingredient is recovered using this non-protein excipient.

Aspects of the present pharmaceutical compositions provide, in part, asugar. As used herein, the term “sugar” refers to a compound comprisingone to 10 monosaccharide units, e.g., a monosaccharide, a disaccharide,a trisaccharide, and an oligosaccharide comprising four to tenmonosaccharide units. It is envisioned that any sugar is useful informulating a Clostridial toxin active ingredient pharmaceuticalcompositions disclosed in the present specification, with the provisothat a therapeutically effective amount of the Clostridial toxin activeingredient is recovered using this sugar. In some embodiments, forexample in a lyophilized composition, the sugar can function as alyoprotector. In some other embodiments, for example in a lyophilizedformulation or in a liquid formulation, the sugar can function as atonicity agent. Monosaccharides are polyhydroxy aldehydes or polyhydroxyketones with three or more carbon atoms, including aldoses, dialdoses,aldoketoses, ketoses and diketoses, as well as cyclic forms, deoxysugars and amino sugars, and their derivatives, provided that the parentmonosaccharide has a (potential) carbonyl group. Monosacchrides includetrioses, like glyceraldehyde and dihydroxyacetone; tetroses, likeerythrose, erythrulose and threose; pentoses, like arabinose, lyxose,ribose, ribulose, xylose, xylulose; hexoses, like allose, altrose,fructose, fucose, galactose, glucose, gulose, idose, mannose, psicose,rhamnose, sorbose, tagatose, talose and trehalose; heptoses, likesedoheptulose and mannoheptulose; octooses, like octulose and2-keto-3-deoxy-manno-octonate; nonoses like sialose; and decose.Oligosaccharides are compounds in which at least two monosaccharideunits are joined by glycosidic linkages. According to the number ofunits, they are called disaccharides, trisaccharides, tetrasaccharides,pentasaccharides, hexoaccharides, heptoaccharides, octoaccharides,nonoaccharides, decoaccharides, etc. An oligosaccharide can beunbranched, branched or cyclic. Common disaccharides include, withoutlimitation, sucrose, lactose, maltose, trehalose, cellobiose,gentiobiose, kojibiose, laminaribiose, mannobiose, melibiose, nigerose,rutinose, and xylobiose. Common trisaccharides include, withoutlimitation, raffinose, acarbose, maltotriose, and melezitose. Othernon-limiting examples of specific uses of sugar excipients can be foundin, e.g., Ansel, supra, (1999); Gennaro, supra, (2000); Hardman, supra,(2001); and Rowe, supra, (2003), each of which is hereby incorporated byreference in its entirety

In an embodiment, a Clostridial toxin active ingredient pharmaceuticalcomposition comprises a sugar. In aspects of this embodiment, aClostridial toxin active ingredient pharmaceutical composition comprisesa monosaccharide. In other aspects of this embodiment, a Clostridialtoxin active ingredient pharmaceutical composition comprises adisaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, ahexoaccharide, a heptoaccharide, an octoaccharide, a nonoaccharide, or adecoaccharide. In yet other aspects of this embodiment, a Clostridialtoxin active ingredient pharmaceutical composition comprises anoligosaccharide comprising two to ten monosaccharide units.

It is envisioned that any amount of sugar is useful in formulating aClostridial toxin active ingredient pharmaceutical compositionsdisclosed in the present specification, with the proviso that atherapeutically effective amount of the Clostridial toxin activeingredient is recovered using this sugar amount. In aspects of thisembodiment, the amount of sugar added to the formulation is about 0.1%(w/w), about 0.5% (w/w), about 1.0% (w/w), about 1.5% (w/w), about 2.0%(w/w), about 2.5% (w/w), about 3.0% (w/w), about 3.5% (w/w), about 4.0%(w/w), about 4.5% (w/w), about 5.0% (w/w), about 5.5% (w/w), about 6.0%(w/w), about 6.5% (w/w), about 7.0% (w/w), about 7.5% (w/w), about 8.0%(w/w), about 8.5% (w/w), about 9.0% (w/w), about 9.5% (w/w), about 10%(w/w), about 15% (w/w), about 20% (w/w), about 25% (w/w), about 30%(w/w), or about 35% (w/w). In other aspects of this embodiment, theamount of sugar added to the formulation is at least 0.1% (w/w), atleast 0.5% (w/w), at least 1.0% (w/w), at least 1.5% (w/w), at least2.0% (w/w), at least 2.5% (w/w), at least 3.0% (w/w), at least 3.5%(w/w), at least 4.0% (w/w), at least 4.5% (w/w), at least 5.0% (w/w), atleast 5.5% (w/w), at least 6.0% (w/w), at least 6.5% (w/w), at least7.0% (w/w), at least 7.5% (w/w), at least 8.0% (w/w), at least 8.5%(w/w), at least 9.0% (w/w), at least 9.5% (w/w), at least 10% (w/w), atleast 15% (w/w), at least 20% (w/w), at least 25% (w/w), at least 30%(w/w), or at least 35% (w/w). In yet other aspects of this embodiment,the amount of sugar added to the formulation is at most 0.1% (w/w), atmost 0.5% (w/w), at most 1.0% (w/w), at most 1.5% (w/w), at most 2.0%(w/w), at most 2.5% (w/w), at most 3.0% (w/w), at most 3.5% (w/w), atmost 4.0% (w/w), at most 4.5% (w/w), at most 5.0% (w/w), at most 5.5%(w/w), at most 6.0% (w/w), at most 6.5% (w/w), at most 7.0% (w/w), atmost 7.5% (w/w), at most 8.0% (w/w), at most 8.5% (w/w), at most 9.0%(w/w), at most 9.5% (w/w), at most 10% (w/w), at most 15% (w/w), at most20% (w/w), at most 25% (w/w), at most 30% (w/w), or at most 35% (w/w).

In an embodiment, the present Clostridial toxin active ingredientpharmaceutical composition comprises a disaccharide. Commondisaccharides include, without limitation, sucrose, lactose, maltose,trehalose, cellobiose, gentiobiose, kojibiose, laminaribiose,mannobiose, melibiose, nigerose, rutinose, and xylobiose. In aspects ofthis embodiment, the Clostridial toxin active ingredient pharmaceuticalcomposition comprises sucrose. In one specific embodiment, theClostridial toxin active ingredient pharmaceutical composition comprisestrehalose. In aspects of this embodiment, the amount of disaccharideadded to the formulation added to the formulation is about 0.1% (w/w),about 0.5% (w/w), about 1.0% (w/w), about 1.5% (w/w), about 2.0% (w/w),about 2.5% (w/w), about 3.0% (w/w), about 3.5% (w/w), about 4.0% (w/w),about 4.5% (w/w), about 5.0% (w/w), about 5.5% (w/w), about 6.0% (w/w),about 6.5% (w/w), about 7.0% (w/w), about 7.5% (w/w), about 8.0% (w/w),about 8.5% (w/w), about 9.0% (w/w), about 9.5% (w/w), about 10% (w/w),about 15% (w/w), about 20% (w/w), about 25% (w/w), about 30% (w/w), orabout 35% (w/w).

Aspects of the present pharmaceutical compositions provide, in part, asurfactant. It is envisioned that any surfactant is useful informulating a Clostridial toxin active ingredient pharmaceuticalcompositions disclosed in the present specification, with the provisothat a therapeutically effective amount of the Clostridial toxin activeingredient is recovered using this surfactant amount. Non-limitingexamples of surfactants include polysorbates like polysorbate 20 (TWEEN®20), polysorbate 40 (TWEEN® 40), polysorbate 60 (TWEEN® 60), polysorbate61 (TWEEN® 61), polysorbate 65 (TWEEN® 65), polysorbate 80 (TWEEN® 80),and polysorbate 81 (TWEEN® 81); poloxamers (polyethylene-polypropylenecopolymers), like Poloxamer 124 (PLURONIC® L44), Poloxamer 181(PLURONIC® L61), Poloxamer 182 (PLURONIC® L62), Poloxamer 184 (PLURONIC®L64), Poloxamer 188 (PLURONIC® F68), Poloxamer 237 (PLURONIC® F87),Poloxamer 338 (PLURONIC® L108), Poloxamer 407 (PLURONIC® F127),polyoxyethyleneglycol dodecyl ethers, like BRIJ® 30, and BRIJ® 35;2-dodecoxyethanol (LUBROl®-PX); polyoxyethylene octyl phenyl ether(TRITON® X-100); sodium dodecyl sulfate (SDS); solutol HS15;3[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); sucrose monolaurate; and sodium cholate. Other non-limitingexamples of surfactant excipients can be found in, e.g., Ansel, supra,(1999); Gennaro, supra, (2000); Hardman, supra, (2001); and Rowe, supra,(2003), each of which is hereby incorporated by reference in itsentirety.

Thus in an embodiment, a Clostridial toxin active ingredientpharmaceutical composition comprises a surfactant. In aspects of thisembodiment, a Clostridial toxin active ingredient pharmaceuticalcomposition comprises a polysorbate, a poloxamer, apolyoxyethyleneglycol dodecyl ether, 2-dodecoxyethanol, polyoxyethyleneoctyl phenyl ether, sodium dodecyl sulfate,3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,3-[(3-Cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulfonate,sucrose monolaurate; or sodium cholate.

It is envisioned that any amount of surfactant is useful in formulatinga Clostridial toxin active ingredient pharmaceutical compositionsdisclosed in the present specification, with the proviso that atherapeutically effective amount of the Clostridial toxin activeingredient is recovered using this surfactant amount. In aspects of thisembodiment, the amount of surfactant added to the formulation is about0.01% (w/w), about 0.02% (w/w), about 0.03% (w/w), about 0.04% (w/w),about 0.05% (w/w), about 0.06% (w/w), about 0.07% (w/w), about 0.08%(w/w), about 0.09% (w/w), about 0.1% (w/w), about 0.5% (w/w), about 1.0%(w/w), about 1.5% (w/w), about 2.0% (w/w), about 2.5% (w/w), about 3.0%(w/w), about 3.5% (w/w), about 4.0% (w/w), about 4.5% (w/w), about 5.0%(w/w), about 5.5% (w/w), about 6.0% (w/w), about 6.5% (w/w), about 7.0%(w/w), about 7.5% (w/w), about 8.0% (w/w), about 8.5% (w/w), about 9.0%(w/w), about 9.5% (w/w), about 10% (w/w), about 15% (w/w), about 20%(w/w), about 25% (w/w), about 30% (w/w), or about 35% (w/w). In otheraspects of this embodiment, the amount of surfactant added to theformulation is at least 0.01% (w/w), at least 0.02% (w/w), at least0.03% (w/w), at least 0.04% (w/w), at least 0.05% (w/w), at least 0.06%(w/w), at least 0.07% (w/w), at least 0.08% (w/w), at least 0.09% (w/w),at least 0.1% (w/w), at least 0.5% (w/w), at least 1.0% (w/w), at least1.5% (w/w), at least 2.0% (w/w), at least 2.5% (w/w), at least 3.0%(w/w), at least 3.5% (w/w), at least 4.0% (w/w), at least 4.5% (w/w), atleast 5.0% (w/w), at least 5.5% (w/w), at least 6.0% (w/w), at least6.5% (w/w), at least 7.0% (w/w), at least 7.5% (w/w), at least 8.0%(w/w), at least 8.5% (w/w), at least 9.0% (w/w), at least 9.5% (w/w), atleast 10% (w/w), at least 15% (w/w), at least 20% (w/w), at least 25%(w/w), at least 30% (w/w), or at least 35% (w/w). In yet other aspectsof this embodiment, the amount of surfactant added to the formulation isat most 0.01% (w/w), at most 0.02% (w/w), at most 0.03% (w/w), at most0.04% (w/w), at most 0.05% (w/w), at most 0.06% (w/w), at most 0.07%(w/w), at most 0.08% (w/w), at most 0.09% (w/w), at most 0.1% (w/w), atmost 0.5% (w/w), at most 1.0% (w/w), at most 1.5% (w/w), at most 2.0%(w/w), at most 2.5% (w/w), at most 3.0% (w/w), at most 3.5% (w/w), atmost 4.0% (w/w), at most 4.5% (w/w), at most 5.0% (w/w), at most 5.5%(w/w), at most 6.0% (w/w), at most 6.5% (w/w), at most 7.0% (w/w), atmost 7.5% (w/w), at most 8.0% (w/w), at most 8.5% (w/w), at most 9.0%(w/w), at most 9.5% (w/w), at most 10% (w/w), at most 15% (w/w), at most20% (w/w), at most 25% (w/w), at most 30% (w/w), or at most 35% (w/w).

In some embodiments, the Clostridial toxin active ingredientpharmaceutical composition comprises a poloxamer. Poloxamers which canbe used with the present pharmaceutical composition include Poloxamer124 (PLURONIC® L44), Poloxamer 181 (PLURONIC® L61), Poloxamer 182(PLURONIC® L62), Poloxamer 184 (PLURONIC® L64), Poloxamer 188 (e.g.,PLURONIC® F68, KOLLIPHOR® P 188), Poloxamer 237 (PLURONIC® F87),Poloxamer 338 (PLURONIC® L108), Poloxamer 407 (PLUIRONIC® F127). In someembodiments, poloxamer 188 may be more advantageous.

In some embodiments, the Clostridial toxin active ingredientpharmaceutical composition comprises a polysorbate. Polysorbates whichcan be used with the present pharmaceutical composition includespolysorbate 20 (TWEEN® 20), polysorbate 40 (TWEEN® 40), polysorbate 60(TWEEN® 60), polysorbate 61 (TWEEN® 61), polysorbate 65 (TWEEN® 65),polysorbate 80 (TWEEN® 80), and polysorbate 81 (TWEEN® 81). In someembodiments, polysorbate 20 may be more advantageous than some otherpolysorbates.

Aspects of the present pharmaceutical compositions provide, in part, atleast an antioxidant. Non-limiting examples of antioxidant include,without limitation, methionine, cysteine, NAC, glutathionine, lipoicacid, sodium metabisulfite, sodium thiosulfate, ascorbic acid, butylatedhydroxyanisole, butylated hydroxytoluene, vitamin E and analogsincluding Trolox C; chelators such as EDTA (ethylene diamine tetraaceticacid sodium salt), EGTA (ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid), DTPA(diethylenetriaminepentaacetic acid), analogs or derivatives thereof;and combinations thereof. In aspects of this embodiment, the amount ofantioxidant added to the formulation ranges from about 0.01% (w/w) toabout 0.10% (w/w).

It is further envisioned that a Clostridial toxin active ingredientpharmaceutical composition disclosed in the present specification canoptionally include, without limitation, other pharmaceuticallyacceptable components (or pharmaceutical components), including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, osmolality adjusting agents, emulsifying agents,sweetening or flavoring agents, and the like. Various buffers and meansfor adjusting pH can be used to prepare a pharmaceutical compositiondisclosed in the present specification, provided that the resultingpreparation is pharmaceutically acceptable. Such buffers include,without limitation, acetate buffers, borate buffers, citrate buffers,phosphate buffers, neutral buffered saline, and phosphate bufferedsaline. It is understood that acids or bases can be used to adjust thepH of a pharmaceutical composition as needed. It is envisioned that anybuffered pH level can be useful in formulating a Clostridial toxinactive ingredient pharmaceutical composition, with the proviso that atherapeutically effective amount of the Clostridial toxin activeingredient is recovered using this effective pH level. In an aspect ofthis embodiment, an effective pH level is at least about pH 5.0, atleast about pH 5.5, at least about pH 6.0, at least about pH 6.5, atleast about pH 7.0 or at about pH 7.5. In another aspect of thisembodiment, an effective pH level is at most about pH 5.0, at most aboutpH 5.5, at most about pH 6.0, at most about pH 6.5, at most about pH 7.0or at most about pH 7.5. In yet another aspect of this embodiment, aneffective pH level is about pH 5.0 to about pH 8.0, an effective pHlevel is about pH 5.0 to about pH 7.0, an effective pH level is about pH5.0 to about pH 6.0, is about pH 5.5 to about pH 8.0, an effective pHlevel is about pH 5.5 to about pH 7.0, an effective pH level is about pH5.5 to about pH 5.0, is about pH 5.5 to about pH 7.5, an effective pHlevel is about pH 5.5 to about pH 6.5.

The pharmaceutical compositions disclosed herein can have a pH ofbetween about 5 and 8 when reconstituted or upon injection. In certainembodiments, the composition will have a pH below 8, such as, forexample, 7.9, or 7.8, or 7.7, or 7.6, or 7.5, or 7.4, or 7.3, or 7.2, or7.1, or 7.0, or 6.9, or 6.8, or 6.7, or 6.6, or 6.5, or 6.4, or 6.3, or6.2, or 6.1, or 6.0, or 5.9, or 5.8, or 5.7, or 5.6, or 5.5, or 5.4, or5.3, or 5.2, or 5.1, or the like. In some embodiments, the pH rangesfrom 5 to 7.

It is envisioned that any concentration of a buffer can be useful informulating a Clostridial toxin active ingredient pharmaceuticalcomposition, with the proviso that a therapeutically effective amount ofthe Clostridial toxin active ingredient is recovered using thiseffective concentration of buffer. In aspects of this embodiment, aneffective concentration of buffer is at least 0.1 mM, at least 0.2 mM,at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, atleast 0.7 mM, at least 0.8 mM, or at least 0.9 mM. In other aspects ofthis embodiment, an effective concentration of buffer is at least 1.0mM, at least 2.0 mM, at least 3.0 mM, at least 4.0 mM, at least 5.0 mM,at least 6.0 mM, at least 7.0 mM, at least 8.0 mM, or at least 9.0 mM.In yet other aspects of this embodiment, an effective concentration ofbuffer is at least 10 mM, at least 20 mM, at least 30 mM, at least 40mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, orat least 90 mM. In still other aspects of this embodiment, an effectiveconcentration of buffer is at least 100 mM, at least 200 mM, at least300 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700mM, at least 800 mM, or at least 900 mM. In further aspects of thisembodiment, an effective concentration of buffer is at most 0.1 mM, atmost 0.2 mM, at most 0.3 mM, at most 0.4 mM, at most 0.5 mM, at most 0.6mM, at most 0.7 mM, at most 0.8 mM, or at most 0.9 mM. In still otheraspects of this embodiment, an effective concentration of buffer is atmost 1.0 mM, at most 2.0 mM, at most 3.0 mM, at most 4.0 mM, at most 5.0mM, at most 6.0 mM, at most 7.0 mM, at most 8.0 mM, or at most 9.0 mM.In yet other aspects of this embodiment, an effective concentration ofbuffer is at most 10 mM, at most 20 mM, at most 30 mM, at most 40 mM, atmost 50 mM, at most 60 mM, at most 70 mM, at most 80 mM, or at most 90mM. In still other aspects of this embodiment, an effectiveconcentration of buffer is at most 100 mM, at most 200 mM, at most 300mM, at most 400 mM, at most 500 mM, at most 600 mM, at most 700 mM, atmost 800 mM, or at most 900 mM. In still further aspects of thisembodiment, an effective concentration of buffer is about 0.1 mM toabout 900 mM, 0.1 mM to about 500 mM, 0.1 mM to about 100 mM, 0.1 mM toabout 90 mM, 0.1 mM to about 50 mM, 1.0 mM to about 900 mM, 1.0 mM toabout 500 mM, 1.0 mM to about 100 mM, 1.0 mM to about 90 mM, or 1.0 mMto about 50 mM.

Embodiments described herein can be practiced with a composition thatcomprises a plurality of botulinum toxin serotypes, such as botulinumtoxin serotypes selected from the group consisting of botulinum toxinserotypes A, B, C₁ D, E, F and G. In certain embodiments, purifiedbotulinum toxins, can be used. In other embodiments, modified botulinumtoxins may be used.

In some embodiments, the composition may optionally also include NaCl.NaCl may particularly preferably be included in compositions comprisingbotulinum toxin, trehalose or sucrose, poloxamer 188, and methionine,and is particularly preferably included in liquid compositionscomprising botulinum toxin, trehalose or sucrose, poloxamer 188, andmethionine. In some lyophilized formulations, NaCl may function as atonicity agent in a reconstitution vehicle. In one embodiment, NaCl maybe present in an amount of 0.9% (w/w) in the reconstitution vehicle.

In some embodiments, the Clostridial toxin active ingredientpharmaceutical composition can be formulated as a lyophilized (i.e.freeze dried) or vacuum dried powder which can be reconstituted with asuitable fluid, such as saline or water, prior to administration to apatient. In alternative embodiments, the pharmaceutical composition canbe formulated as an aqueous solution or suspension.

In some embodiments, the solid Clostridial toxin active ingredientpharmaceutical composition comprises a botulinum toxin, a tonicity agentand/or a lyoprotector, a poloxamer and/or a polysorbate and anantioxidant. In some embodiments, the Clostridial toxin activeingredient pharmaceutical composition comprises a botulinum toxin. Insome embodiments, the Clostridial toxin active ingredient pharmaceuticalcomposition comprises trehalose. In some embodiments, the Clostridialtoxin active ingredient pharmaceutical composition comprises poloxamer188 or polysorbate 20. In some embodiments, the composition comprisesEDTA EGTA, DTPA, or analogs thereof. In alternative embodiments, thecomposition comprises methionine and/or NAC. In aspects of thesealternative embodiments, the composition further comprises EDTA, EGTA,DTPA, or analogs thereof. In some embodiments, the composition furthercomprises a buffering agent. In one embodiment, the compositioncomprises histidine buffer. In some embodiments, the relative weightamounts of disaccharide, poloxamer and antioxidant are within thefollowing ranges: trehalose: 1 to 15%, 1 to 10%, or 2-15% or 2-10%;poloxamer: 0.5-8% or 0.5 to 5%; methionine: 0.01 to 5%, 0.02 to 3%, 0.05to 1%, 0.05 to 0.5%. In some embodiments, the relative weight amounts oftrehalose, poloxamer and methionine are within the following ranges:trehalose: 1 to 15%, 1 to 10%, or 2-15% or 2-10%; poloxamer: 0.5-8% or0.5 to 5%; methionine: 0.01 to 5%, 0.02 to 3%, 0.05 to 1%, 0.05 to 0.5%.In some embodiments, the relative weight amounts of trehalose, poloxamerand methionine are within the following ranges respectively: 1 to 10%;0.5 to 5% and 0.1 to 0.3%. In other embodiments, the relative weightamounts of trehalose, polysorbate and methionine are within thefollowing ranges respectively: 1 to 15%; 0.02% to 0.06%; and 0.1 to0.3%. In other embodiments, the relative weight amounts of trehalose,polysorbate and methionine are within the following ranges respectively:1 to 10%; 0.02% to 0.06%; and 0.1 to 0.3%. In some embodiments, therelative weight amount of EDTA or an EDTA analog is from about 0.01 to0.10%. In some embodiments, the relative weight amount of NAC rangesfrom 0.01 to 0.5%.

In aspects of these embodiments, the Clostridial toxin active ingredientpharmaceutical composition is formulated as a solid (i.e., lyophilizedor vacuum dried) composition. In some embodiments, the solid Clostridialpharmaceutical composition comprises a lyoprotector. In someembodiments, the preferred lyoprotector includes sucrose, trehalose,mannitol, sorbitol, glucose, or combinations thereof. In someembodiments, the solid pharmaceutical composition comprises NAC in arelative weight amount of 0.01 to 0.5%. In some embodiments, thepharmaceutical composition further comprises EDTA, EGTA, DTPA, oranalogs thereof. In alternative embodiments, the solid pharmaceuticalcomposition comprises methionine and EDTA, EGTA, DTPA, or analogsthereof.

In one embodiment, the composition is a solid composition consisting ofbotulinum toxin type A, 8% (w/w) trehalose, 4% (w/w) poloxamer 188, 0.2%(w/w) methionine, and a Histidine buffer. In one embodiment, the solidcomposition is reconstituted with a reconstitution vehicle comprisingNaCl prior to administration to a patient. In one embodiment, NaCl maybe present in an amount of 0.9% (w/w) in the reconstitution vehicle.

In an alternative aspect of these embodiments, the Clostridial toxinactive ingredient pharmaceutical composition is formulated as a liquid.In some embodiments, the liquid pharmaceutical composition comprises atonicity agent selected from trehalose, sucrose, sodium chloride,mannitol, sorbitol, glucose, and combinations thereof. In someembodiments, the liquid pharmaceutical composition comprises NAC in arelative weight amount of 0.1 to 0.5%. In some embodiments, the liquidpharmaceutical composition comprises NAC and a chelating agent selectedfrom EDTA, EGTA, DTPA, and analogs thereof. In some embodiments, theliquid pharmaceutical composition comprises histidine buffer. In someembodiments, the liquid pharmaceutical composition has a pH from 5 to 7.

Methods of Treatment

In embodiments, methods of treating diseases, disorders, conditions, andthe like, are described which comprise administering a pharmaceuticalcomposition as described herein to a subject in need thereof in anamount sufficient to produce improved patient function. In certainembodiments, the diseases are of a neuromuscular nature, such as, forexample, those diseases that affect muscles and nerve control thereof,such as, for example, overactive bladder, and the like. Certainembodiments relate to the treatment of pain, such as, for example,treatment of headache pain, or back pain, or muscle pain, or the like.In certain embodiments, methods encompass the treatment of psychologicaldisorders, including, for example, depression, anxiety, and the like.

Compositions and methods described herein can be useful for thetreatment, reduction of symptoms, and/or prevention of, for example,achalasia, anal fissure, anismus, blepharospasm, cerebral palsy,cervical dystonia, cervicogenic headache, hemifacial spasm, dyshidroticeczema, dysphagia, dysphonia, esophageal dysmotility, esophagealmuscular ring, esotropia (infantile), eyelift, facial myokemia, gaitdisturbances (idiopathic toe-walking), generalized dystonia, hemifacialspasm, hyperfunctional facial lines (glabellar, forehead, crows' feet,down-turned angles of the mouth), hyperhidrosis, incontinence(idiopathic or neurogenic), medication overuse headache, migraineheadache, myoclonus, muscle mass or activity reduction, involving, forexample, the masseter or the like, myofascial pain syndrome, obstructiveurinary symptoms, pancreas divisum pancreatitis, Parkinson's disease,puborectalis syndrome, reduction of surgical scar tension, salivaryhypersecretion, sialocele, sixth nerve palsy, spasticity, speech/voicedisorders, strabismus, surgery adjunct (ophthalmic), tardive dyskinesia,temporomandibular joint disorders, tension headache, thoracic outletsyndrome, torsion dystonia, torticolis, Tourette's syndrome, tremor,whiplash-associated neck pain, pain, itching, inflammation, allergy,cancer and benign tumors, fever, obesity, infectious diseases, viral andbacterial, hypertension, cardiac arrhythmias, vasospasm,atherosclerosis, endothelial hyperplasia, venous thrombosis, varicoseveins, apthous stomatitis, hypersalivation, temporomandibular jointsyndrome, hyperhidrosis, bromhidrosis, acne, rosacea, hyperpigmention,hypertrophic scars, keloids, calluses and corns, skin wrinkling,excessive sebum production, psoriasis, dermatitis, allergic rhinitis,nasal congestion, post nasal drip, sneezing, ear wax, serous andsuppurative otitis media, tonsil and adenoid hypertrophy, tinnitus,dizziness, vertigo, hoarseness, cough, sleep apnea, snoring, glaucoma,conjunctivitis, uveitis, strabismus, Grave's disease, excessive hairgrowth, hair loss, asthma, bronchitis, emphysema, mucus production,pleuritis, coagulation disorders, myeloproliferative disorders,disorders involving eosinophils, neutrophils, macrophages andlymphocytes, immune tolerance and transplantation, autoimmune disorders,dysphagia, acid reflux, hiatal hernia, gastritis and hyperacidity,diarrhea and constipation, hemorrhoids, urinary incontinence, prostatichypertrophy, erectile dysfunction, priapism and Peyronie's disease,epididymitis, contraception, menstrual cramps, preventing prematuredelivery, endometriosis and fibroids, arthritis, osteoarthritis,rheumatoid, bursitis, tendonitis, tenosynovitis, fibromyalgia, seizuredisorders, spasticity, headache, and neuralgias.

In certain embodiments, patients are limited to a maximum of 360 U ofbotulinum toxin administered over any 90-day period.

Treatment of Nerve/Muscle Conditions

In an embodiment, the neuromuscular disease is hyperhidrosis. A subjectsuffering from hyperhidrosis, for example, receives about 59 U peraxilla, or about 58 U per axilla, or about 57 U per axilla, or about 56U per axilla, or about 55 U per axilla, or about 54 U per axilla, orabout 53 U per axilla, or about 52 U per axilla, or about 51 U peraxilla, or about 50 U per axilla, or about 49 U per axilla, or about 48U per axilla, or about 47 U per axilla, or about 46 U per axilla, orabout 45 U per axilla, or about 44 U per axilla, or about 43 U peraxilla, or about 42 U per axilla, or about 41 U per axilla, or about 40U per axilla, or about 39 U per axilla, or about 38 U per axilla, orabout 37 U per axilla, or about 36 U per axilla, or less, per treatmentof a pharmaceutical composition described herein. In an embodiment, 50 Utotal are injected intradermally into 10-15 sites spaced approximately1-2 cm apart.

In an embodiment, the neuromuscular disease is hemifacial spasm. Asubject suffering from hemifacial spasm, for example receives betweenabout 1.5 to 15 U per treatment of a pharmaceutical compositiondescribed herein. In a further example, the subject receives betweenabout 1.5 to 3 U, 1.5 to 5 U, 1.5 to 7 U, 1.5 to 10 U, 1.5 to 12 U, 1.5to 15 U, 5 to 10 U, 5 to 15 U, or 10 to 15 U per treatment areadministered to a patient with hemifacial spasm. In a still furtherexample, the subject receives about 1.5 U, about 2 U, about 2.5 U, about3 U, about 3.5 U, about 4 U, about 4.5 U about 5 U, about 5.5 U, about 6U, about 6.5 U, about 7 U, about 7.5 U, about 8 U, about 8.5 U, about 9U, about 9.5 U, about 10 U, about 10.5 U, about 11 U, about 11.5 U,about 12 U, about 12.5 U, about 13 U, about 13.5 U, about 14 U, about14.5 U, or about 15 U per treatment are administered to a patient withhemifacial spasm. Dosages greater than 15 U per treatment may also beadministered to patients with hemifacial spasm to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the neuromuscular disease is cervical dystonia. Asubject suffering from cervical dystonia, for example, receives betweenabout 15 to 300 U per treatment of a pharmaceutical compositiondescribed herein. In a further example, the subject receives betweenabout 35 to 250 U, 65 to 200 U, 85 to 175 U, 105 to 160 U, or 125 to 145U are administered to a patient with cervical dystonia. In anembodiment, dosages to the sternocleidomastoid muscle is limited to 100U or less. Dosages greater than 300 U per treatment may also beadministered to patients with cervical dystonia to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the neuromuscular disease is blepharospasm. A subjectsuffering from blepharospasm, for example, receives between about 1.25to 2.5 U of a pharmaceutical composition described herein injected intothe medial and lateral pretarsal orbicularis oculi of the upper lid andinto the lateral pretarsal orbicularis oculi of the lower lid. In afurther example, the subject receives about 1.5 U, about 1.6 U, about1.7 U, about 1.8 U, about 1.9 U, about 2.0 U, about 2.1 U, about 2.2 U,about 2.3 U, about 2.4 U, about 2.5 U, or more, per injection site. Atreatment session can comprise multiple treatments.

In an embodiment, the neuromuscular disease is strabismus. A subjectsuffering from strabismus, for example, receives between about 1.25 to2.5 U per injection site of a pharmaceutical composition describedherein. In a further example, the subject receives about 1.5 U, about1.6 U, about 1.7 U, about 1.8 U, about 1.9 U, about 2.0 U, about 2.1 U,about 2.2 U, about 2.3 U, about 2.4 U, about 2.5 U, or more, perinjection site to achieve a therapeutic response. In embodiments, lowerdoses are used for treatment of small deviations. In embodiments,vertical muscles and horizontal strabismus of less than 20 prismdiameters can be treated with 1.25 to 2.5 U per injection site. Atreatment session can comprise multiple treatments.

In an embodiment, the neuromuscular disease is muscle spasticity. Asubject suffering from muscle spasticity, for example, receives betweenabout 20 to 200 U per treatment of a pharmaceutical compositiondescribed herein. In a further example, the subject receives betweenabout 20 to 30 U, 20 to 40 U, 20 to 60 U, 20 to 80 U, 20 to 100 U, 20 to125 U, 20 to 150 U, or 20 to 175 U per treatment are administered to apatient with muscle spasticity. In a still further example, the subjectreceives about 20 U, about 25 U, about 30 U, about 35 U, about 40 U,about 45 U, about 50 U, about 55 U, about 60 U, about 65 U, about 70 U,about 75 U, about 80 U, about 85 U, about 90 U, about 95 U, about 100 U,about 105 U, about 110 U, about 115 U, about 120 U, about 125 U, about130 U, about 135 U, about 140 U, about 145 U, about 150 U, about 155 U,about 160 U, about 165 U, about 170 U, about 175 U, about 180 U, about185 U, about 190 U, about 195 U, or about 200 U per treatment areadministered to a patient with muscle spasticity. In an embodiment, thebiceps brachii can be injected with between 100 U and 200 U divided into4 injection sites. In an embodiment, the flexor carpi radialis can beinjected with between 12.5 U and 50 U in 1 injection site. In anembodiment, the flexor carpi ulnaris can be injected with between 12.5 Uand 50 U in 1 injection site. In an embodiment, the flexor digitorumprofundus can be injected with between 30 U and 50 U in one injectionsite. In an embodiment, the flexor digitorum sublimis can be injectedwith between 30 U and 50 in a single injection site. Dosages greaterthan 200 U per treatment may also be administered to patients withmuscle spasticity to achieve a therapeutic response. A treatment sessioncan comprise multiple treatments.

Treatment of Pain

In another embodiment, methods for treating pain comprising the step ofadministering a pharmaceutical composition described herein to a subjectin need thereof in an amount sufficient to reduce pain. In anotherembodiment, the patient suffers from myofascial pain, migraine headachepain, tension headache pain, neuropathic pain, facial pain, lower-backpain, sinus-headache pain, pain associated with temporomandibular jointdisease, pain associated with spasticity or cervical dystonia,post-surgical wound pain, or neuralgia. A treatment session can comprisemultiple treatments.

In an embodiment, the patient suffers from facial pain. A subjectsuffering from facial pain, for example, receives between about 4 to 40U per treatment of a pharmaceutical composition described herein. In afurther example, the subject receives between about 4 to 10 U, 4 to 15U, 4 to 20 U, 4 to 25 U, 4 to 30 U, 4 to 35 U, 7 to 15 U, 7 to 20 U, 7to 25 U, 7 to 30 U, 7 to 35 U, or 7 to 40 U per treatment areadministered to a patient suffering from facial pain. In a still furtherexample, the subject receives about 4 U, about 5 U, about 7.5 U, about10 U, about 12.5 U, about 15 U, about 17.5 U, about 20.0 U, about 22.5U, about 25.0 U, about 27.5 U, about 30.0 U, about 32.5 U, about 35 U,about 37.5 U, or about 40 U per treatment are administered to a patientwith facial pain. Dosages greater than 40 U per treatment may also beadministered to patients with facial pain to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the patient suffers from myofascial pain. A subjectsuffering from myofascial pain, for example, receives between about 5 to100 U per treatment of a pharmaceutical composition described herein. Ina further example, the subject receives between about 5 to 10 U, 5 to 20U, 5 to 30 U, 5 to 40 Units, 5 to 50 Units, 5 to 60 Units, 5 to 70Units, 5 to 80 Units, 5 to 90 U, 10 to 20 U, 10 to 30 U, 10 to 50 U, or10 to 60 U, or 10 to 70 U, or 10 to 80 U, 10 to 90 U, or 10 to 100 U pertreatment are administered to a patient suffering from myofascial pain.In a further example, the subject receives about 5 U, about 10 U, about15 U, about 20 U, about 25 U, about 30 U, about 35 U, about 40 U, about45 U, about 50 U, about 55 U, about 60 U, about 65 U, about 70 U, about75 U, about 80 U, about 85 U, about 90 U, about 95 U, or about 100 U pertreatment. Dosages greater than 100 U per treatment may also beadministered to patients with myofascial pain to achieve a therapeuticresponse. A treatment session can comprise multiple treatments.

In an embodiment, the subject suffers from lower-back pain. A subjectsuffering from lower-back pain, for example, receives between about 15to 150 U per treatment of a pharmaceutical composition described herein.In a further example, the subject receives between about 15 to 30 U, 15to 50 U, 15 to 75 U, 15 to 100 U, 15 to 125 U, 15 to 150 U, 20 to 100 U,20 to 150 U, or 100 to 150 U per treatment. In a still further example,the subject receives about 15 U, about 20 U, about 25 U, about 30 U,about 35 U, about 40 U, about 45 U, about 50 U, about 55 U, about 60 U,about 65 U, about 70 U, about 75 U, about 80 U, about 85 U, about 90 U,about 95 U, about 100 U, about 105 U, about 110 U, about 115 U, about120 U, about 125 U, about 130 U, about 135 U, about 140 U, about 145 U,or about 150 U per treatment to alleviate lower-back pain. Dosagesgreater than 150 U per treatment may also be administered to patientswith lower-back pain to achieve a therapeutic response. A treatmentsession can comprise multiple treatments.

In an embodiment, the patient suffers from migraine headache pain,including wherein the patient suffers from migraine headaches of 4 hoursor more 15 or more days per month. A subject suffering frommigraine-headache pain, for example, receives between about 0.5 to 200 Uper treatment of a pharmaceutical composition described herein. In afurther example, the subject receives between about 5 to 190 U, 15 to180 U, 25 to 170 U, 35 to 160 U, 45 to 150 U, 55 to 140 U, 65 to 130 U,75 to 120 U, 85 to 110 U, or 95 to 105 U per treatment to alleviatemigraine headache pain. A treatment session can comprise multipletreatments.

For example, about 0.5 U, about 1.0 U, about 1.5 U, about 2.0 U, about2.5 U, about 3.0 U, about 3.5 U, about 4.0 U, about 4.5 U, about 5.0 U,about 5.5 U, about 6.0 U, about 6.5 U, about 7.0 U, about 7.5 U, about8.0 U, about 8.5 U, about 9.0 U, about 9.5 U, about 10.0 U, about 12 U,about 15 U, about 17 U, about 20 U, about 22 U, about 25 U, about 27 U,about 30 U, about 32 U, about 35 U, about 37 U, about 40 U, about 42 U,about 45 U, about 47 U, or about 50 U per treatment site areadministered to a patient with migraine-headache pain. A patient can betreated at multiple sites, such as, for example, 2 sites, 3 sites, 4sites, 5 sites, 6 sites, 7 sites, 8 sites, 9 sites, 10 sites, 11 sites,12 sites, 13 sites, 14 sites, 15 sites, 16 sites, 17 sites, 18 sites, 19sites, 20 sites, 21 sites, 22 sites, 23 sites, 24 sites, 25 sites, 26sites, 27 sites, 28 sites, 29 sites, 30 sites, 31 sites, 32 sites, ormore, or the like. In an embodiment, a patient suffering from migraineis injected 31 times with 5 U per 0.1 mL injection, across thecorrugator (2 injections of 5 U each), procerus (1 injection of 5 U),frontalis (4 injections of 5 U each), temporalis (8 injections of 5 Ueach), occipitalis (6 injections of 5 U each), cervical paraspinal (4injections of 5 U each), and trapezius (6 injections of 5 U each)muscles. With the exception of the procerus muscle which can be injectedat the midline, all muscles can, in certain embodiments, be injectedbilaterally with half of the injection sites to the left and half to theright side of the head and neck. Dosages greater than 200 U pertreatment may also be administered to patients with migraine-headachepain to achieve a therapeutic response. A treatment session can comprisemultiple treatments.

In an embodiment, the patient suffers from sinus-headache pain. Asubject suffering from sinus-headache pain, for example, receivesbetween about 4 to 40 U per treatment of a pharmaceutical compositiondescribed herein. In a further example, the subject receives betweenabout 4 to 10 U, 4 to 15 U, 4 to 20 U, 4 to 25 U, 4 to 30 U, 4 to 35 U,7 to 15 U, 7 to 20 U, 7 to 25 U, 7 to 30 U, 7 to 35 U, or 7 to 40 U pertreatment to alleviate sinus-headache pain. In a still further example,the subject receives about 4 U, about 5 U, about 7.5 U, about 10 U,about 12.5 U, about 15 U, about 17.5 U, about 20.0 U, about 22.5 U,about 25.0 U, about 27.5 U, about 30.0 U, about 32.5 U, about 35 U,about 37.5 U, or about 40 U per treatment. Dosages greater than 40 U pertreatment may also be administered to patients with sinus headache-painto achieve a therapeutic response. A treatment session can comprisemultiple treatments.

In an embodiment, the patient suffers from tension-headache pain. Asubject suffering from tension-headache pain, for example, receivesbetween about 5 to 50 U per treatment of a pharmaceutical compositiondescribed herein. In a further example, between about 5 to 10 U, 5 to 15U, 5 to 20 U, 5 to 25 U, 5 to 30 U, 5 to 35 U, 5 to 40 U, 5 to 45 U, 10to 20 U, 10 to 25 U, 10 to 30 U, 10 to 35 U, 10 to 40 U, or 10 to 45 Uper treatment are administered to a patient with tension-headache pain.In a still further example, the subject receives about 5 U, about 10 U,about 20 U, about 25 U, about 30 U, about 35 U, about 40 U, about 45 U,or about 50 U per treatment administered to alleviate tension-headachepain. In an embodiment, a patient suffering from tension headache isinjected 31 times with 5 U per 0.1 mL injection, across the corrugator(2 injections of 5 U each), procerus (1 injection of 5 U), frontalis (4injections of 5 U each), temporalis (8 injections of 5 U each),occipitalis (6 injections of 5 U each), cervical paraspinal (4injections of 5 U each), and trapezius (6 injections of 5 U each)muscles. With the exception of the procerus muscle which can be injectedat the midline, all muscles can, in certain embodiments, be injectedbilaterally with half of the injection sites to the left and half to theright side of the head and neck. Dosages greater than 200 U pertreatment may also be administered to patients with tension headachepain to achieve a therapeutic response. A treatment session can comprisemultiple treatments.

In an embodiment, the patient suffers from sinus headache pain or facialpain associated with acute or recurrent chronic sinusitis. For example,a pharmaceutical composition as described herein can be administered tothe nasal mucosa or to the subcutaneous structures overlying thesinuses, wherein the administration of the formulation reduces theheadache and/or facial pain associated with acute recurrent or chronicsinusitis. In further embodiments, any of the pharmaceuticalformulations described herein can be administered to the nasal mucosa orto the subcutaneous structures overlying the sinuses, such as over oneor more of the sinuses selected from the group consisting of: ethmoid;maxillary; mastoid; frontal; and sphenoid. In another embodiment,subcutaneous structures overlying the sinuses lie within one or more ofthe areas selected from the group consisting of: forehead; malar;temporal; post auricular; and lip. In embodiments, multiple injectionsof 5 U each are administered to treat the sinus headache pain or facialpain associated with acute or recurrent chronic sinusitis.

In another embodiment, a patient suffering from sinus headache pain orfacial pain associated with acute or recurrent chronic sinusitis istreated by administering any of the pharmaceutical formulationsdescribed herein to an afflicted area of the patient. In a furtherembodiment, the pharmaceutical formulations disclosed herein areadministered to the projections of a trigeminal nerve innervating asinus.

Patients suffering from sinus headache pain or facial pain associatedwith acute or recurrent chronic sinusitis often exhibit symptomsincluding rhinitis, sinus hypersecretion and/or purulent nasaldischarge. In one embodiment, patients treated with the pharmaceuticalcompositions described herein exhibit, prior to treatment, symptoms ofsinus hypersecretion and purulent nasal discharge.

Embodiments contemplated herein provide methods for treating a patientsuffering from sinus headache pain or facial pain associated with acuteor recurrent chronic sinusitis, wherein the subject suffers fromneuralgia. In certain embodiments, the neuralgia is trigeminalneuralgia. In another embodiment, the neuralgia is: associated withcompressive forces on a sensory nerve; associated with intrinsic nervedamage, demyelinating disease, or a genetic disorder; associated with ametabolic disorder; associated with central neurologic vascular disease;or associated with trauma. In another embodiment, the pain is associatedwith dental extraction or reconstruction.

Treatment of Urological Disorders

In an embodiment, methods for treating a patient suffering fromoveractive bladder (OAB), such as, for example, that due to a neurologiccondition (NOAB), or idiopathic OAB (IOAB) are provided. For example,pharmaceutical formulations described herein can be administered to thebladder or its vicinity, e.g. the detrusor, wherein the administrationof the formulation reduces the urge incontinence associated withoveractive bladder. In certain embodiments, the dosage can be, forexample, 200 U, or more, or less, or the like. For example, the dosagecan be about 15 U, about 20 U, about 25 U, about 30 U, about 35 U, about40 U, about 45 U, about 50 U, about 55 U, about 60 U, about 65 U, about70 U, about 75 U, about 80 U, about 85 U, about 90 U, about 95 U, about100 U, about 105 U, about 110 U, about 115 U, about 120 U, about 125 U,about 130 U, about 135 U, about 140 U, about 145 U, about 150 U, about160 U, about 170 U, about 180 U, about 190 U, about 200 U, about 210 U,about 220, about 230 U, about 240 U, or more, or the like, pertreatment. A patient can be injected at multiple sites, such as, forexample, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites,9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites, 18 sites, 19 sites, 20 sites, 21 sites, 22 sites, 23sites, 24 sites, 25 sites, 26 sites, 27 sites, 28 sites, 29 sites, 30sites, 31 sites, 32 sites, 33 sites, 34 sites, 35 sites, 36 sites, 37sites, 38 sites, or more, or the like. In an embodiment, patientssuffering from OAB are treated with 30 1 mL injections of approximately6.7 U per injection into the detrusor muscle.

In an embodiment, methods for treating a patient suffering fromneurogenic detrusor overactivity (NDO), such as that due to a neurologiccondition, are provided. For example, pharmaceutical formulations can beadministered to the bladder or its vicinity, e.g. the detrusor, whereinthe administration of the formulation reduces the urge incontinenceassociated with overactive bladder. In certain embodiments, the dosagecan be, for example, 200 U, or more, or less, or the like. For example,the dosage can be about 15 U, about 20 U, about 25 U, about 30 U, about35 U, about 40 U, about 45 U, about 50 U, about 55 U, about 60 U, about65 U, about 70 U, about 75 U, about 80 U, about 85 U, about 90 U, about95 U, about 100 U, about 105 U, about 110 U, about 115 U, about 120 U,about 125 U, about 130 U, about 135 U, about 140 U, about 145 U, about150 U, about 160 U, about 170 U, about 180 U, about 190 U, about 200 U,about 210 U, about 220, about 230 U, about 240 U, or more, or the like,per treatment. A patient can be injected at multiple sites, such as, forexample, 2 sites, 3 sites, 4 sites, 5 sites, 6 sites, 7 sites, 8 sites,9 sites, 10 sites, 11 sites, 12 sites, 13 sites, 14 sites, 15 sites, 16sites, 17 sites, 18 sites, 19 sites, 20 sites, 21 sites, 22 sites, 23sites, 24 sites, 25 sites, 26 sites, 27 sites, 28 sites, 29 sites, 30sites, 31 sites, 32 sites, or more, or the like. In an embodiment,patients suffering from NDO are treated with 30 1 mL injections ofapproximately 6.7 U per injection into the detrusor muscle.

Treatment of Cosmetic Features

In another embodiment, methods for cosmetically modifying soft-tissuefeatures comprising the step of administering at least onepharmaceutical composition as described herein to a subject in needthereof in an amount sufficient to modify said features are provided. Ina further embodiment, the pharmaceutical composition is administered viatranscutaneous or transmucosal injection either at a single focus ormultiple foci.

In embodiments, pharmaceutical formulations are administered to the faceor neck of the subject. In a further embodiment, the pharmaceuticalformulations are administered to the subject in an amount sufficient toreduce rhytides. For example, the formulation can be administeredbetween eyebrows of the subject in an amount sufficient to reducevertical lines between the eyebrows and on a bridge of a nose. Thepharmaceutical formulations can also be administered near either one orboth eyes of the subject in an amount sufficient to reduce lines atcorners of the eyes. In an embodiment, compositions can be injectedlocally to smooth skin. In another embodiment, the pharmaceuticalformulations can also be administered to a forehead of the subject in anamount sufficient to reduce horizontal lines on said forehead. In yetanother embodiment, the pharmaceutical formulation is administered tothe neck of the subject in an amount sufficient to reduce muscle bandsin the neck. In an embodiment, a pharmaceutical composition is appliedto the masseter muscle to relax the muscle and/or decrease massetermass.

In a further embodiment, the patient suffers from facial wrinkles. Asubject suffering from facial wrinkles, for example, can receive betweenabout 1 to 100 U per treatment of a pharmaceutical formulation. In afurther example, the subject receives between about 1 to 10 U, 1 to 20U, 1 to 30 U, 1 to 40 U, 1 to 50 U, 1 to 60 U, 1 to 70 U, 1 to 80 U, 1to 90 U, 5 to 20 U, 5 to 30 U, 5 to 40 U, 5 to 50 U, 5 to 60 U, 5 to 70U, 5 to 80 U, 5 to 90 U, or 5 to 100 U per treatment. In a still furtherexample, the subject receives about 1 U, about 10 U, about 20 U, about30 U, about 40 U, about 50 U, about 60 U, about 70 U, about 80 U, about90 U, or about 100 U per treatment. Dosages greater than 100 U pertreatment may also be administered to patients suffering frominflammation or an inflammatory disorder to achieve a therapeuticresponse.

Treatment of Inflammation

In another embodiment, methods for treating inflammation comprising thestep of administering a pharmaceutical composition as described hereinto a subject in need thereof in an amount sufficient to reduceinflammation. In certain embodiments, pharmaceutical formulations areadministered to a patient without producing muscle weakness. In anembodiment, the pharmaceutical formulations are administered to patientswith an inflammatory condition. In certain embodiments, the inflammatorycondition is neurogenic inflammation. In another embodiment, the subjectsuffers from rheumatoid arthritis or a gastro-intestinal inflammatorydisease.

In a further embodiment, the patient suffers from an inflammatorydisorder. A subject suffering from an inflammatory disorder, forexample, receives between about 1 to 100 U per treatment of apharmaceutical composition as described herein. In a further example,the subject receives between about 1 to 10 U, 1 to 20 U, 1 to 30 U, 1 to40 U, 1 to 50 U, 1 to 60 U, 1 to 70 U, 1 to 80 U, 1 to 90 U, 5 to 20 U,5 to 30 U, 5 to 40 U, 5 to 50 U, 5 to 60 U, 5 to 70 U, 5 to 80 U, 5 to90 U, or 5 to 100 U per treatment. In a still further example, thesubject receives about 1 U, about 10 U, about 20 U, about 30 U, about 40U, about 50 U, about 60 U, about 70 U, about 80 U, about 90 U, or about100 U per treatment. Dosages greater than 100 U per treatment may alsobe administered to patients suffering from inflammation or aninflammatory disorder to achieve a therapeutic response.

Treatment of Skin Conditions

A method for treating a skin disorder can have the step of localadministration of a botulinum neurotoxin to a location of a skindisorder of a patient, such as to a face, hand or foot of a patient. Theneurotoxin can be locally administered in an amount of between about10⁻³ units/kg of patient weight and about 35 units/kg of patient weight.For example, the neurotoxin is locally administered in an amount ofbetween about 10⁻² U/kg and about 25 U/kg of patient weight. In afurther example, the neurotoxin is administered in an amount of betweenabout 10⁻¹ U/kg and about 15 U/kg. In one method, the neurotoxin islocally administered in an amount of between about 1 U/kg and about 10U/kg in a composition as described herein. In a clinical setting, it canbe advantageous to administer from 1 U to 3000 U of a neurotoxin, suchas botulinum toxin type A or B, to a skin disorder location by topicalapplication or by subdermal administration, to effectively treat theskin disorder.

Administration of botulinum toxin can be carried out at multiple sitesin the skin, wherein the sites of adjacent injections are separated byabout 0.1 to 10 cm, or about 0.5 to about 5 cm, for example, by about1.5 to about 3 cm. The toxins may be any of the botulinum toxins A, B,C, D, E, F, G or a mosaic toxin. The amounts administered may varybetween 0.1 and 1000 U, or about 1 to about 40, or from about 5 to about10 U, depending on the manufactures specifications, the class of thetoxin and the mode of administration. The repeat time range for theseadministrations for maintenance of the desired change variessubstantially according to the location of the injection, the conditionto be adjusted and the condition of the patient. Thus, the repeat timemay vary from about 1 week to about 50 weeks, however, a common range isabout 4 to about 25 weeks, or even about 12 weeks to about 16 weeks.

The distances between administration sites, such as, for example,injection sites, can vary from about 1 mm to about 10 cm, suitably fromabout 5 mm to about 5 cm, and more usually from about 1 cm to about 3cm. Thus, for example botulinum A may be suitably administered byintradermal injection between about 0.1 to about 10 U at a separation offrom about 0.5 to about 10 cm.

In another embodiment, methods for treating cutaneous disorderscomprising the step of administering a pharmaceutical composition asdescribed herein to a subject in need thereof in an amount sufficient toreduce a sebaceous or mucous secretion is provided. In furtherembodiments, the pharmaceutical compositions as described herein areadministered to a patient without producing muscle weakness. In certainembodiments, the pharmaceutical composition as described herein areinjected into one or more sites of an eyelid or conjunctiva. In anotherembodiment, the formulations are administered to a body surface.

In another embodiment, the pharmaceutical formulations are administeredin an amount sufficient to reduce cutaneous bacterial or fungal growth,including but not limited to Staphylococcus; Streptococcus andMoraxella. For example, the pharmaceutical compositions as describedherein are administered to an area selected from the group consistingof: eyelid; scalp; feet; groin; and armpit to reduce cutaneousinfection.

Treatment of Depression

In another embodiment, a method for treating depression is provided.Depression is a general term for recognized forms of depression that aredefined and are separately diagnosed according to criteria given inhandbooks for psychiatry, for example in the Diagnostic and StatisticalManual of Mental Disorders 4th edition (DSM-IV) published by theAmerican Psychiatric Association, Washington, D.C. (1994). In theDSM-IV, depressive disorders are classified under mood disorders and aredivided into three types: major depressive disorder, dysthymic disorderand depressive disorder not otherwise specified (or “atypical”). Ingeneral, regardless of whether or not the depressive syndrome ismelancholic, atypical, or some admixture of the two, a diagnosis ofmajor depression is given when depressed mood is present, or loss ofinterest or pleasure in all activities is present, for at least twoweeks.

Depression is often associated with psychomotor abnormalities, such asincreased or retarded motor activity. Many depressed persons can also berecognized by their “depressed facies” in which the muscles of facialexpression assume a distressed or sad appearance. For example, the browmay be furrowed, the inner ends of the eyebrows raised, and the anglesof the mouth lowered such that the facial appearance is recognizably sadand/or anxious. Four major muscle groups are involved in frowning: thefrontal, procerus, corrugator supercilii and orbicularis oculi (Weideret al. Derm Surg. 24:1172-1174, 1998. The corrugator supercilii is alsoknown as the “scowl” muscle.

A subject diagnosed with depression or experiencing a depressive episodeis treated by administering any of the pharmaceutical compositionsdescribed herein. In a further embodiment, the pharmaceuticalformulations disclosed herein are administered to the patient viainjection subcutaneously. Example 10 describes treatment of a personwith a form of depression by administering a therapeutically effectiveamount of botulinum toxin in a composition comprising botulinum toxin,trehalose, a poloxamer surfactant, and methionine. The composition isadministered to a facial muscle involved in frowning or scowling. Theneurotoxin affects the ability of the subject to frown and/or scowl,thereby treating depression. More generally, a therapeutically effectiveamount of Botulinum toxin A can be injected into one or more of thefrontalis, procerus, the corrugator supercilii, orbicularis oculi, orthe depressor anguli oris (trianglaris muscle).

In another embodiment, the method comprises administering atherapeutically effective amount of a Clostridial toxin activeingredient in a composition as described herein to a facial muscleinvolved in frowning, scowling, or a sad appearance. The Clostridialtoxin active ingredient causes partial or complete paralysis of thefacial muscle, thereby affecting the ability of the subject to frownand/or scowl, or appear sad, and thereby treat depression. For example,a therapeutically effective amount of a composition comprising theClostridial toxin active ingredient botulinum toxin, along with asurfactant, an antioxidant, and optionally a tonicity modifier, can beinjected into one or more of the orbicularis oculi, frontalis, procerus,the corrugator supercilii, or the depressor anguli oris (trianglarismuscle). In a specific example, the composition comprising Botulinumtoxin A is injected into the procerus muscle over the glabella. Otheradministration points and paradigms are disclosed, for example, in U.S.Pat. No. 7,758,872, which is incorporated by reference herein.

In other embodiments of the method, adult subjects with moderate tosevere major depressive disorder (MDD), either single episode orrecurrent are contemplated for treatment, where the MDD diagnosis isbased upon the DSM-IV-TR criteria. In one embodiment, a single treatmentis contemplated, and in other embodiments, a single, repeated treatmentis contemplated, with the treatment is repeated at intervals of 2-6,2-4, or 3-6 months. The amount of Clostridial toxin active ingredientdosed is, for example, 30 U or 50 U, where, in some embodiments, thedose is divided into a plurality of injections. In one embodiment, theplurality of injections is 6 and in another embodiment, is 8. In oneembodiment, 30 U is divided into 6 injections to the glabellar region ofthe forehead (procerus and corrugator muscles). In one embodiment, 50 Uis divided into 8 injections to the glabellar region of the forehead(procerus and corrugator muscles).

Effective treatment is indicated by, for example, a primary efficacymeasure known in the art, such as the clinical assessment known asMontgomery-Asberg Depression Rating scale. Additional efficacy measures:clinic CGI-S score (Clinical Global Impression of Change scores), clinicHAM-D17 total score (Hamilton Rating Scale for Depression).

Treatment of Cardiac Arrhythmia

In another embodiment, a method for treating cardiac arrhythmia isprovided. Arrhythmias are caused by a disruption of the normalfunctioning of the electrical conduction system of the heart. Normally,the chambers of the heart (atria and ventricles) contract in acoordinated manner. The signal to contract is an electrical impulse thatbegins in the sinoatrial nod, and the impulse is conducted through theatria and stimulates them to contract. The impulse passes through theatrioventricular node, then travels through the ventricles andstimulates them to contract. Problems can occur anywhere along theconduction system, causing various arrhythmias. Problems can also occurin the heart muscle itself, causing it to respond differently to thesignal to contract, also causing arrhythmias, or causing the ventriclesto contract independently of the normal conduction system.

Arrhythmias include tachycardias, bradycardias and true arrhythmias ofdisturbed rhythm. Arrhythmias are classified as lethal if they cause asevere decrease in the pumping function of the heart. When the pumpingfunction is severely decreased for more than a few seconds, bloodcirculation is essentially stopped, and organ damage (such as braindamage) can occur within a few minutes. Lethal arrhythmias includeventricular fibrillation, also ventricular tachycardia that is rapid andsustained, or pulseless, and may include sustained episodes of otherarrhythmias. Additional types of arrhythmias include atrial fibrillationor flutter, multifocal atrial tachycardia, paroxysmal supraventriculartachycardia, Wolff-Parkinson-White syndrome, sinus tachycardia, sinusbradycardia, bradycardia associated with heart block, sick sinussyndrome, and ectopic heartbeat.

Accordingly, a method for treating cardiac arrhythmia is provided, themethod comprising the step of administering a composition as describedherein that comprises a therapeutically effective amount of Clostridialtoxin active ingredient, the composition administered locally to theheart of a patient with a cardiac arrhythmia or at risk of a cardiacarrhythmia. Particular arrhythmias treatable include bradycardia andtachycardia. In one embodiment, the composition is locally administered,by which is meant administration directly to, in, or to the vicinity of,the cardiac muscle to be treated. Local administration includesintrapericardial, intracardiac cardiac catheterization and directcardiac muscle injection routes of administration for the composition.

Example 11 describes treatment of a person undergoing cardiac surgery byadministering a therapeutically effective amount of botulinum toxin in acomposition comprising botulinum toxin, trehalose, a poloxamersurfactant, and NAC. In one embodiment, the composition is administeredvia injection into one or more epicardial fat pads of the heart. Thedose administered, in one exemplary embodiment, 25 U per epicardial fatpad, to a total dose of 125 U. In another exemplary embodiment, 50 U perepicardial fat pad, to a total dose of 250 U, is administered.

Effective treatment is indicated by, for example, a primary efficacyendpoint of, for example, incidence of atrial fibrillation (AF) asmeasured by ECG for 4 weeks or at 4 weeks post treatment. Additionalefficacy endpoints include length of hospital stay, length of stay inICU, rehospitalization rate, anticoagulant medication use, need forinterventional procedures for post operative atrial fibrillation, suchas ablation, pacemaker implantation, electrical or pharmacologiccardioversion.

EXAMPLES

The following examples illustrate embodiments and aspects of the presentcompositions and methods and are not intended to limit the scopethereof.

Example 1 Activities and Stabilities of Exemplary Solid ClostridialPharmaceutical Compositions

Bulk solutions of botulinum toxin were prepared by mixing an appropriatealiquot of a botulinum toxin type A with a solution having theingredients set forth in Tables 1-3. Four formulations were prepared fortesting, referred to as Formulation 1, Formulation 2, Formulation 3 andFormulation 4, and three comparative formulations were prepared,referred to as Comparator 1, Comparator 2, and Comparator 3.Formulations 1, 2, 3 and 4 were composed of a disaccharide, asurfactant, an antioxidant and a histidine buffer. Each formulation wasfilled into a glass vial and lyophilized using conventionalfreeze-drying conditions. Portions of the lyophilized formulations werestored under several conditions; for example, at 25° C. and at 40° C.for six months (Tables 1.1 and 1.2), or at −20° C. and 40° C. for 1month (Table 2), or at 25° C. for 7.5 months (Table 3). Potency of thelyophilized formulations after storage and reconstitution with salinewas tested by a cell based potency assay, as described in U.S. Pat. No.8,618,261, the assay details of which are incorporated by referenceherein. The results were normalized to target. The potencies of thesolid compositions are shown in Tables 1-3.

Example 2 Activities of Liquid Clostridial Pharmaceutical Compositionsin the Presence or Absence of Antioxidants

Bulk drug product solutions were prepared by mixing an appropriatealiquot of a botulinum toxin type A with three different vehiclesolutions as shown in Table 4. All three formulations contained 8% w/wtrehalose, 4% w/w poloxamer P188 and 20 mM histidine buffer at pH 6.0.Formulation 10 contained no antioxidant. Formulations 11 and 12contained NAC and methionine, respectively. The bulk solutions werefilled into 2 mL glass vials (1.25 mL fill), and sealed with rubberstopper and aluminum shell. Potency of the formulations was tested by acell based potency assay after filling (time zero, t0) and after storagefor one month at four temperatures (−70° C., 5° C., 25° C. and 40° C.).The target potency was 100 Units/mL. Potency test results are given inTable 4.

Example 3 Impact of Exemplary Antioxidants on the Stability of ExemplaryLiquid Formulations

Bulk drug product solutions were prepared by mixing an appropriatealiquot of the BoNT/A drug substance with different antioxidants asshown in Table 3.1. All compositions contained 8% w/w trehalose, 4% w/wpoloxamer P188, 20 mM histidine buffer at pH 6.0 and 0.2 wt % of one ormore of the following antioxidants: NAC, L-methionine, L-tryptophan,L-glutathione, sodium sulfite, and/or propyl gallate, and/or EDTA. Theformulations are specified in Table 3.1. Target potency was 100 U/mL.The bulk solutions were filled into 2 mL glass vials (1.25 mL fill), andsealed with rubber stopper and aluminum shell. Potency of theformulations was tested by a cell based potency assay after filling(time zero, t0) and after storage at 40° C. for 2 weeks and 1 month.Potency test results are given in Table 5, above.

TABLE 3.1 Formulation Toxin Tre P188 NAC Met TRP GSH NaSul PrpGal EDTANo. U/mL % % Buffer % % % % % % % Formulation 100 8 4 20 mM 0.2 20 His,pH 6.0 Formulation 100 8 4 20 mM 0.2 21 His, pH 6.0 Formulation 100 8 420 mM 0.2 22 His, pH 6.0 Formulation 100 8 4 20 mM 0.2 23 His, pH 6.0Formulation 100 8 4 20 mM 0.2 24 His, pH 6.0 Formulation 100 8 4 20 mM0.2 0.2 25 His, pH 6.0 Formulation 100 8 4 20 mM 0.2 0.03 26 His, pH 6.0Formulation 100 8 4 20 mM 0.2 0.2 0.03 27 His, pH 6.0 Formulation 100 84 20 mM 0.2 0.2 28 His, pH 6.0 Tre = trehalose, P188 = poloxamer P188;His = L-histidine; NAC = N-acetyl-L-cysteine; Met = L-methionine; TRP =L-tryptophan; GSH = L-glutathione; NaSul = sodium sulfite; PrpGal =propyl gallate; EDTA = ethylene diamine tetraacetic acid, sodium salt.

Example 4 Impact of Exemplary Disaccharides on the Stability ofExemplary Liquid Compositions

Liquid compositions were prepared by mixing an appropriate aliquot ofBoNT/A (100 U/mL) with solutions of one of 8% w/w trehalose or 8% w/wsucrose, and of 4% w/w poloxamer P188 and 0.2 w/w % L-methionine, in 20mM histidine buffer at pH 6.0. The composition with trehalose wasassigned Formulation No. 30 and the composition with sucrose wasassigned Formulation No. 31. The compositions were stored at 25° C. andpotency was tested by cell based potency assay prior to storage (timezero, t0) and at selected time points after storage. Potency testresults are shown in Table 6.

Example 5 Impact of Exemplary Surfactants on the Stability of ExemplaryLiquid Compositions

Liquid compositions were prepared by mixing an appropriate aliquot ofBoNT/A (100 U/mL) with solutions comprising poloxamer P188 (4 w/w %) orpolysorbate (TWEEN® 20, 0.04 w/w %) and 8% w/w trehalose, 0.2 w/w %L-methionine, in 20 mM histidine buffer at pH 6.0. The composition withpoloxamer P188 was identified as Formulation No. 30 and the compositionwith polysorbate was identified as Formulation No. 32. Potency wastested by cell based potency assay prior to storage (time zero, t0) andafter 1 month of storage at 40° C. Potency test results are shown inTable 7.

Example 6 Stability of Toxin in Compositions with No Tonicity Agent

Five liquid compositions were prepared by mixing an appropriate aliquotof BoNT/A (100 U/mL or 150 U/mL) with solutions comprising or lackingpoloxamer P188 (4 w/w %), 8% w/w trehalose, and 0.2 w/w % L-methionine,in 20 mM histidine buffer at pH 6.0. Details of the compositions aregiven in Table 6.1:

TABLE 6.1 Poloxamer Formulation Toxin Trehalose P188 Methionine No. U/mL% % % Buffer Formulation 100 8 4 0 20 mM His, 33 pH 6.0 Formulation 1500 0 0.2 20 mM His, 34 pH 6.0 Formulation 150 8 0 0.2 20 mM His, 35 pH6.0 Formulation 100 0 4 0.2 20 mM His, 36 pH 6.0 Formulation 100 8 4 0.220 mM His, 37 pH 6.0

Potency of Formulations 33-37 was tested by cell based potency assayprior to storage (time zero, t0) and after 1 month of storage at −70° C.and 40° C. for 1 month. Potency test results are shown in Table 8.

Example 7 Stability of Toxin in Liquid Compositions

Liquid compositions were prepared by mixing an appropriate aliquot ofBoNT/A (100 U/mL) with solutions comprising poloxamer P188 (4 w/w %) orpolysorbate (TWEEN®-20, 0.04 w/w %), sucrose or trehalose (8% w/w) orNaCl (0.9 w/w %), and 0.2 w/w % L-methionine, in water or 20 mMhistidine buffer at pH 6.0. Details of the compositions are given inTable 7.1:

TABLE 7.1 Polysorbate Toxin Treh Suc NaCl P188 (TWEEN ®20) Met Buffer*Formulation No. U/mL % % % % % % or water Formulation 31 100 8 4 0.2buffer Formulation 38 100 8 4 0.2 water Formulation 39 100 0.9 4 0.2buffer Formulation 40 100 8 0.04 0.2 buffer Tre = trehalose; Suc =sucrose; P188 = poloxamer P188; Met = L-methionine *Buffer = 20 mM His,pH 6.0

Potency of Formulations 31 and 38-40 was tested by a cell based potencyassay prior to storage (time zero, t0) and after 5.5 months, 7.5 monthsand 12 months of storage at 5° C. Potency test results are shown inTable 9.

Example 8 Stability of Toxin in Liquid Compositions with No TonicityAgent

Liquid compositions were prepared by mixing an appropriate aliquot ofBoNT/A (200 U/mL) with solutions comprising poloxamer P188 (4 w/w %) orpolysorbate (TWEEN®-20, 0.04 w/w %) and 0.2 w/w % L-methionine, in 20 mMhistidine buffer at pH 6.0. Details of the compositions are given inTable 8.1.

TABLE 8.1 Polysorbate Formulation Toxin P188 (TWEEN ® methionine No.U/mL % 20) % % Buffer Formulation 200 0 0.04 0.2 20 mM His, 41 pH 6.0Formulation 200 4 0 0.2 20 mM His, 42 pH 6.0 P-188 = poloxamer P188; Met= L-methionine

Potency of Formulations 41 and 42 was tested by a cell based potencyassay prior to storage (time zero, t0) and 2 weeks of storage at −70° C.and 40° C. Potency test results are shown in Table 10.

Example 9 Impact of Exemplary Lyoprotectors and Surfactants on theStability of Exemplary Lyophilized Formulations

Lyophilized compositions were prepared as follows. Bulk solutions ofbotulinum toxin were prepared by mixing an appropriate aliquot of abotulinum toxin type A (BoNT/A, 200 U/mL) with solutions comprisingpoloxamer P188 (4 w/w %) or polysorbate (TWEEN® 20, 0.04 w/w %), withtrehalose or sucrose (8 w/w %), and 0.2 w/w % L-methionine, in 20 mMhistidine buffer at pH 6.0. Details of the compositions are given inTable 9.1. The solutions were filled into glass vials and lyophilizedusing conventional freeze-drying conditions.

TABLE 9.1 Toxin Target Poloxamer Formulation Potency Trehalose SucroseTWEEN ®20 P188 Methionine No. U/vial % % % % % Buffer Formulation 200 80 4 0.2 20 mM 43 His, pH 6.0 Formulation 200 8 0.04 0 0.2 20 mM 44 His,pH 6.0 Formulation 200 0 8 0 4 0.2 20 mM 45 His, pH 6.0 Formulation 2000 8 0.04 0 0.2 20 mM 46 His, pH 6.0

Potency of the lyophilized compositions, designated as Formulations43-46, was tested by a cell based potency assay after reconstitution ofthe lyophiles subsequent to storage for 2 weeks at −20° C. and 40° C.Potency test results are shown in Table 12.

Example 10 Method for Treating Depression

A lyophilized composition comprised of botulinum toxin type A (BoNT/A,200 U), 8 wt % trehalose, 4 wt % poloxamer P188, 0.2 wt % methionine, in20 mM histidine buffer, pH 6.0, is prepared as set forth in Example 9.The lyophilized composition is reconstituted in saline prior toadministration.

A 50-year-old male reports a history of depression for two years. He ispresently taking antidepressant medications (PAXIL® 20 mg orally per dayand WELLBUTRIN 225 mg orally per day). The male subject reportscontinued depression. He is treated by injecting into thefrown/glabellar lines 30 Unit equivalents of the reconstitutedcomposition. Four months later he reports a much-improved mood with nofeelings of depression. His frown/glabellar lines are re-treated with 30Unit equivalents of the reconstituted composition.

Example 11 Method for Treating Cardia Arrhythmia

A formulation similar to Formulation 1 as described in Example 1 isprepared. The lyophilized composition is comprised of botulinum toxintype A (BoNT/A, 50 U), 2 wt % trehalose, 4 wt % poloxamer P188, 0.3 wt %NAC, in 20 mM histidine buffer, pH 5.5. The lyophilized composition isreconstituted in saline prior to administration.

A Caucasian male is undergoing cardiac surgery. As part of the surgicalprocedure, an amount of the reconstituted composition to provide a totaldose of 75 U botulinum toxin is evenly divided for administration viainjection into three epicardial fat pads of his heart. The medical staffreports no arrhythmia during or post-surgery.

Many alterations and modifications may be made by those having ordinaryskill in the art, without departing from the spirit and scope of thedisclosure. Therefore, it must be understood that the describedembodiments have been set forth only for the purposes of examples, andthat the embodiments should not be taken as limiting the scope of thefollowing claims. The following claims are, therefore, to be read toinclude not only the combination of elements which are literally setforth, but all equivalent elements for performing substantially the samefunction in substantially the same way to obtain substantially the sameresult. The claims are thus to be understood to include those that havebeen described above, those that are conceptually equivalent, and thosethat incorporate the ideas of the disclosure.

What is claimed is:
 1. A liquid pharmaceutical composition, comprising:(i) a Clostridial toxin active ingredient; (ii) a tonicity agentselected from trehalose, sucrose, sodium chloride, mannitol, sorbitol,glucose, and combinations thereof; (iii) a surfactant selected from apoloxamer, a polysorbate, and combinations thereof; and (iv) anantioxidant selected from methionine, N-acetyl cysteine,ethylenediaminetetraacetic acid, and combinations thereof.
 2. The liquidpharmaceutical composition of claim 1, wherein the Clostridial toxinactive ingredient is a botulinum toxin.
 3. The liquid pharmaceuticalcomposition of claim 2, wherein the tonicity agent is trehalose.
 4. Theliquid pharmaceutical composition of claim 3, wherein trehalose ispresent in an amount between about 1 to 15 wt %.
 5. The liquidpharmaceutical composition of claim 2, wherein the surfactant is apoloxamer.
 6. The liquid pharmaceutical composition of claim 5, whereinthe poloxamer is present in an amount between about 0.5 to 8 wt %. 7.The liquid pharmaceutical composition of claim 2, wherein theantioxidant is methionine.
 8. The liquid pharmaceutical composition ofclaim 7, wherein methionine is present in an amount between about 0.01to 0.5 wt %.
 9. The liquid pharmaceutical composition of claim 2,wherein the composition comprises trehalose, poloxamer 188 andmethionine.
 10. The liquid pharmaceutical composition of claim 2,wherein the antioxidant comprises N-acetyl cysteine.
 11. A liquidpharmaceutical composition, comprising: (i) a Clostridial toxin activeingredient; (ii) trehalose in an amount between about 1-15 wt %; (iii)poloxamer in an amount between about 0.5-8 wt %; and (iv) methionine inan amount between about 0.05-5 wt %.
 12. The liquid pharmaceuticalcomposition of claim 11, wherein the Clostridial toxin active ingredientis a botulinum toxin.
 13. The liquid pharmaceutical composition of claim11, wherein methionine is present in an amount between about 0.05-2 wt%.
 14. The liquid pharmaceutical composition of claim 11, wherein thepoloxamer is present in an amount between about 2-6 wt %.
 15. The liquidpharmaceutical composition of claim 11, wherein trehalose is present inan amount between about 5-12 wt %.
 16. A lyophilized pharmaceuticalcomposition, comprising: (i) a Clostridial toxin active ingredient; (ii)a lyoprotector selected from trehalose, sucrose, mannitol, sorbitol,glucose, and combinations thereof; (iii) a surfactant selected from apoloxamer, a polysorbate, and combinations thereof; and (iv) anantioxidant selected from methionine, N-acetyl cysteine,ethylenediaminetetraacetic acid, and combinations thereof.
 17. Thelyophilized pharmaceutical composition of claim 16, wherein theClostridial toxin active ingredient is a botulinum toxin.
 18. Thelyophilized pharmaceutical composition of claim 16, wherein thelyoprotector is trehalose.
 19. The lyophilized pharmaceuticalcomposition of claim 18, wherein trehalose is present in an amountbetween about 1 to 10 wt %.
 20. The lyophilized pharmaceuticalcomposition of claim 16, wherein the surfactant is a poloxamer.
 21. Thelyophilized pharmaceutical composition of claim 20, wherein thepoloxamer is present in an amount between about 0.5 to 5 wt %.
 22. Thelyophilized pharmaceutical composition of claim 16, wherein theantioxidant is methionine.
 23. The lyophilized pharmaceuticalcomposition of claim 22, wherein methionine is present in an amountbetween about 0.01 to 0.5 wt %.
 24. The lyophilized pharmaceuticalcomposition of claim 16, wherein the composition comprises trehalose,poloxamer 188 and methionine.
 25. The lyophilized pharmaceuticalcomposition of claim 24, wherein the composition comprises 8 w/w %trehalose, 4 w/w % poloxamer 188 and 0.2 w/w % methionine.
 26. Thelyophilized pharmaceutical composition of claim 16, wherein theantioxidant comprises N-acetyl cysteine.
 27. A method for treatingdepression, comprising: providing for administration a composition ofclaim
 16. 28. A method for treating cardiac arrhythmia, comprising:providing for administration a composition of claim
 16. 29. Alyophilized pharmaceutical composition, comprising: (i) a Clostridialtoxin active ingredient; (ii) trehalose, sucrose, mannitol, sorbitol,glucose, or combinations thereof; (iii) a poloxamer; and (iv)methionine.
 30. The lyophilized pharmaceutical composition of claim 29,wherein the Clostridial toxin active ingredient is a botulinum toxin.31. The lyophilized pharmaceutical composition of claim 30, whereintrehalose, when present, is in an amount between 1-10 wt % and sucrose,when present, is in an amount between 0.5-5 wt %.
 32. The lyophilizedpharmaceutical composition of claim 30, wherein the poloxamer is presentin an amount between about 0.5 to 5 wt %.
 33. The lyophilizedpharmaceutical composition of claim 30, wherein the methionine ispresent in an amount between 0.05-5 wt %.
 34. A method for treatingdepression, comprising: providing for administration a composition ofclaim
 29. 35. A method for treating cardiac arrhythmia, comprising:providing for administration a composition of claim 29.